Normalizing chromosomes for the determination and verification of common and rare chromosomal aneuploidies

ABSTRACT

The present invention provides a method capable of detecting single or multiple fetal chromosomal aneuploidies in a maternal sample comprising fetal and maternal nucleic acids, and verifying that the correct determination has been made. The method is applicable to determining copy number variations (CNV) of any sequence of interest in samples comprising mixtures of genomic nucleic acids derived from two different genomes, and which are known or are suspected to differ in the amount of one or more sequence of interest. The method is applicable at least to the practice of noninvasive prenatal diagnostics, and to the diagnosis and monitoring of conditions associated with a difference in sequence representation in healthy versus diseased individuals.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of United Kingdom Patent ApplicationNumber 1106394.8 filed on Apr. 14, 2011, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention provides a method capable of determining single ormultiple fetal chromosomal aneuploidies in a maternal sample comprisingfetal and maternal nucleic acids, and verifying that the correctdetermination has been made. The method is applicable at least to thepractice of noninvasive prenatal diagnostics, and to the diagnosis andmonitoring of conditions associated with a difference in sequencerepresentation in healthy versus diseased individuals.

BACKGROUND OF THE INVENTION

The American College of Obstetrics and Gynecology (ACOG) PracticeBulletin Number 77 published in 2007 supports that first trimesteraneuploidy risk assessment, based on nuchal translucency measurement andsurrogate biochemical markers to screen for Down syndrome, for allpregnant women (ACOG Practice Bulletin No. 77, Obstet Gynecol109:217-227 [2007]). These screening tests can only provide a riskdetermination that is inconclusive and has non-optimal determination andhigh false positive rates. Today, only invasive methods includingchorionic villus sampling (CVS), amniocentesis or cordocentesis providedefinite genetic information about the fetus, but these procedures areassociated with risks to both mother and fetus (Odibo et al., ObstetGynecol 112:813-819 [2008]; Odibo et al., Obstet Gynecol 111:589-595[2008]; Evans and Wapner, Semin Perinatol 29:215-218 [2005]). Therefore,a non-invasive means to obtain definite information on fetal chromosomalstatus is desirable.

Massively parallel DNA sequencing of cfDNA obtained from the maternalplasma yields millions of short sequence tags that can be aligned anduniquely mapped to sites from a reference human genome, and the countingof the mapped tags can be used to determine the over- orunder-representation of a chromosome (Fan et al., Proc Natl Acad Sci USA105:16266-16271 [2008]; Voelkerding and Lyon, Clin Chem 56:336-338[2010]). However, the depth of sequencing and subsequent countingstatistics determines the sensitivity of determination for fetalaneuploidy. The requirement for an optimized algorithm to determinechromosomal aneuploidies in maternal plasma samples is underscored bythe apparent inability to determine more than one type of trisomy in apopulation of test samples (Chiu et al., BMJ 342, c7401 [2011]; Ehrichet al., Am J Obstet Gynecol 2014:205 e1 [2011]).

The limitations of the existing methods underlie the need for optimalnoninvasive methods that would provide any or all of the specificity,sensitivity, and applicability to reliably diagnose chromosomalaneuploidies for prenatal diagnoses and for the diagnoses and monitoringof medical conditions associated with copy number changes.

The present invention fulfills some of the above needs and in particularoffers an advantage in providing a reliable method having sufficientsensitivity to determine single or multiple chromosomal aneuploidies,and which verifies that the correct determination is made.

SUMMARY OF THE INVENTION

The present invention provides a method capable of determining single ormultiple fetal chromosomal aneuploidies in a maternal sample comprisingfetal and maternal nucleic acids, and verifying that the correctdetermination has been made. The method is applicable to determiningcopy number variations (CNV) of any sequence of interest in samplescomprising mixtures of genomic nucleic acids derived from two differentgenomes, and which are known or are suspected to differ in the amount ofone or more sequence of interest. The method is applicable at least tothe practice of noninvasive prenatal diagnostics, and to the diagnosisand monitoring of conditions associated with a difference in sequencerepresentation in healthy versus diseased individuals.

In one embodiment, the method determines the presence or absence of afetal chromosomal aneuploidy in a maternal test sample comprising fetaland maternal nucleic acid molecules by: (a) obtaining sequenceinformation for the fetal and maternal nucleic acids in the maternalsample to identify a number of sequence tags for a chromosome ofinterest and a number of sequence tags for at least two normalizingchromosomes; (b) using the number of sequence tags to calculate a firstand a second normalizing value for the chromosome of interest; and (c)comparing the first normalizing value for the chromosome of interest toa first threshold value and comparing the second normalizing value forthe chromosome of interest to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. The first andsecond threshold values can be the same or they can be different. Instep (c) of this method, the comparison of the first normalizing valuefor said chromosome of interest to a threshold value indicates thepresence or absence of an aneuploidy for said chromosome of interest,and the comparison of the second normalizing value for said chromosomeof interest to a threshold value verifies the determination of thepresence or absence of an aneuploidy for the chromosome of interest. Insome embodiments, the first normalizing value is a first chromosomedose, which is a ratio of the number of sequence tags for the chromosomeof interest and a first normalizing chromosome, and the secondnormalizing value is a second chromosome dose, which is a ratio of thenumber of sequence tags for the chromosome of interest and a secondnormalizing chromosome. Optionally, the first and second normalizingvalues can be expressed as normalized chromosome values (NCV) asdescribed below.

In the above and all subsequent embodiments, the step of obtainingsequencing information comprises next generation sequencing (NGS). NGScan be sequencing-by-synthesis using reversible dye terminators.Alternatively, NGS can be sequencing sequencing-by-ligation. NGS canalso be single molecule sequencing.

Similarly, in the above and all subsequent embodiments, the normalizingchromosomes for chromosome 21 are selected from chromosomes 9, 11, 14,and 1. In some embodiments, the normalizing chromosomes for chromosome18 are selected from chromosomes 8, 3, 2, and 6. In some embodiments,the normalizing chromosomes for chromosome 13 are selected fromchromosome 4, the group of chromosomes 2-6, chromosome 5, and chromosome6. In some embodiments, the normalizing chromosomes for chromosome X areselected from chromosomes 6, 5, 13, and 3. In some embodiments, thenormalizing chromosomes for chromosome 1 are selected from chromosomes10, 11, 9 and 15. In some embodiments, the normalizing chromosomes forchromosome 2 are selected from chromosomes 8, 7, 12, and 14. In someembodiments, the normalizing chromosomes for chromosome 3 are selectedfrom chromosomes 6, 5, 8, and 18. In some embodiments, the normalizingchromosomes for chromosome 4 are selected from chromosomes 3, 5, 6, and13. In some embodiments, the normalizing chromosomes for chromosome 5are selected from chromosomes 6, 3, 8, and 18. In some embodiments, thenormalizing chromosomes for chromosome 6 are selected from chromosomes5, 3, 8, and 18. In some embodiments, the normalizing chromosomes forchromosome 7 are selected from chromosomes 12, 2, 14 and 8. In someembodiments, the normalizing chromosomes for chromosome 8 are selectedfrom chromosomes 2, 7, 12, and 3. In some embodiments, the normalizingchromosomes for chromosome 9 are selected from chromosomes 11, 10, 1,and 14. In some embodiments, the normalizing chromosomes for chromosome10 are selected from chromosomes 1, 11, 9, and 15. In some embodiments,the normalizing chromosomes for chromosome 11 are selected fromchromosomes 1, 10, 9, and 15. In some embodiments, the normalizingchromosomes for chromosome 12 are selected from chromosomes 7, 14, 2,and 8. In some embodiments, the d normalizing chromosomes for chromosome14 are selected from chromosomes 12, 7, 2, and 9. In some embodiments,the normalizing chromosomes for chromosome 15 are selected fromchromosomes 1, 10, 11, and 9. In some embodiments, the normalizingchromosomes for chromosome 16 are selected from chromosomes 20, 17, 15,and 1. In some embodiments, the normalizing chromosomes for chromosome17 are selected from chromosomes 16, 20, 19 and 22. In some embodiments,the normalizing chromosomes for chromosome 19 are selected from 22, 17,16, and 20. In some embodiments, the normalizing chromosomes forchromosome 20 are selected from chromosomes 16, 17, 15, and 1. In someembodiments, the normalizing chromosomes for chromosome 22 are selectedfrom chromosomes 19, 17, 16, and 20.

In another embodiment, the method determines the presence or absence ofa fetal chromosomal aneuploidy in a maternal test sample comprisingfetal and maternal nucleic acid molecules by: (a) obtaining sequenceinformation for the fetal and maternal nucleic acids in the maternalsample to identify a number of sequence tags for a chromosome ofinterest and a number of sequence tags for at least two normalizingchromosomes; (b) using the number of sequence tags to calculate a firstand a second normalizing value for the chromosome of interest; and (c)comparing the first normalizing value for the chromosome of interest toa first threshold value and comparing the second normalizing value forthe chromosome of interest to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. The first andsecond threshold values can be the same or they can be different. Instep (c) of this method, the comparison of the first normalizing valuefor said chromosome of interest to a threshold value indicates thepresence or absence of an aneuploidy for said chromosome of interest,and the comparison of the second normalizing value for said chromosomeof interest to a threshold value verifies the determination of thepresence or absence of an aneuploidy for the chromosome of interest. Insome embodiments, the first normalizing value is a first chromosomedose, which is a ratio of the number of sequence tags for the chromosomeof interest and a first normalizing chromosome, and the secondnormalizing value is a second chromosome dose, which is a ratio of thenumber of sequence tags for the chromosome of interest and a secondnormalizing chromosome. Optionally, the first and second normalizingvalues can be expressed as normalized chromosome values (NCV) asdescribed below. The fetal chromosomal aneuploidy can be a partial or acomplete chromosomal aneuploidy. In these embodiments, the fetalchromosomal aneuploidy can be selected from trisomy 21 (T21), trisomy 18(T18), trisomy 13 (T13), monosomy X. In some embodiments, the maternalsample is obtained from a pregnant woman. In some embodiments, thematernal sample is a biological fluid sample e.g. a blood sample or theplasma fraction derived therefrom. In some embodiments, the maternalsample is a plasma sample. In some embodiments, the nucleic acids in thematernal sample are cfDNA molecules. In some other embodiments, thematernal test sample is a plasma sample obtained from a pregnant womanand the nucleic acid molecules are cfDNA molecules.

In another embodiment, the method determines the presence or absence ofat least two different chromosomal aneuploidies. In one embodiment, themethod determines the presence or absence of at least two differentfetal chromosomal aneuploidies in a maternal test sample comprisingfetal and maternal nucleic acid molecules by repeating the steps (a)-(c)for at least two chromosomes of interest, wherein the steps comprise (a)obtaining sequence information for the fetal and maternal nucleic acidsin the maternal sample to identify a number of sequence tags for achromosome of interest and a number of sequence tags for at least twonormalizing chromosomes; (b) using the number of sequence tags tocalculate a first and a second normalizing value for the chromosome ofinterest; and (c) comparing the first normalizing value for thechromosome of interest to a first threshold value and comparing thesecond normalizing value for the chromosome of interest to a secondthreshold value to determine the presence or absence of a fetalaneuploidy in the sample. The first and second threshold values can bethe same or they can be different. In step (c) of this method, thecomparison of the first normalizing value for said chromosome ofinterest to a threshold value indicates the presence or absence of ananeuploidy for said chromosome of interest, and the comparison of thesecond normalizing value for said chromosome of interest to a thresholdvalue verifies the determination of the presence or absence of ananeuploidy for the chromosome of interest. In some embodiments, thefirst normalizing value is a first chromosome dose, which is a ratio ofthe number of sequence tags for the chromosome of interest and a firstnormalizing chromosome, and the second normalizing value is a secondchromosome dose, which is a ratio of the number of sequence tags for thechromosome of interest and a second normalizing chromosome. Optionally,the first and second normalizing values can be expressed as normalizedchromosome values (NCV) as described herein. In some embodiments, themethod comprises repeating the method for all chromosomes to determinethe presence or absence of at least two different fetal chromosomalaneuploidies.

In another embodiment, the method determines the presence or absence ofat least two different chromosomal aneuploidies. In one embodiment, themethod determines the presence or absence of at least two differentfetal chromosomal aneuploidies in a maternal test sample comprisingfetal and maternal nucleic acid molecules by repeating the steps (a)-(c)for at least two chromosomes of interest, wherein the steps comprise (a)obtaining sequence information for the fetal and maternal nucleic acidsin the maternal sample to identify a number of sequence tags for achromosome of interest and a number of sequence tags for at least twonormalizing chromosomes; (b) using the number of sequence tags tocalculate a first and a second normalizing value for the chromosome ofinterest; and (c) comparing the first normalizing value for thechromosome of interest to a first threshold value and comparing thesecond normalizing value for the chromosome of interest to a secondthreshold value to determine the presence or absence of a fetalaneuploidy in the sample. The first and second threshold values can bethe same or they can be different. In step (c) of this method, thecomparison of the first normalizing value for said chromosome ofinterest to a threshold value indicates the presence or absence of ananeuploidy for said chromosome of interest, and the comparison of thesecond normalizing value for said chromosome of interest to a thresholdvalue verifies the determination of the presence or absence of ananeuploidy for the chromosome of interest. In some embodiments, thefirst normalizing value is a first chromosome dose, which is a ratio ofthe number of sequence tags for the chromosome of interest and a firstnormalizing chromosome, and the second normalizing value is a secondchromosome dose, which is a ratio of the number of sequence tags for thechromosome of interest and a second normalizing chromosome. Optionally,the first and second normalizing values can be expressed as normalizedchromosome values (NCV) as described herein. In some embodiments, themethod comprises repeating the method for all chromosomes to determinethe presence or absence of at least two different fetal chromosomalaneuploidies. The at least two different fetal chromosomal aneuploidiescan be selected from T21, T18, T13, and monosomy X. In some embodiments,the maternal sample is obtained from a pregnant woman. In someembodiments, the maternal sample is a biological fluid sample e.g. ablood sample or the plasma fraction derived therefrom. In someembodiments, the maternal sample is a plasma sample. In someembodiments, the nucleic acids in the maternal sample are cfDNAmolecules. In some other embodiments, the maternal test sample is aplasma sample obtained from a pregnant woman and the nucleic acidmolecules are cfDNA molecules.

In another embodiment, the method verifies the determination of thepresence or absence of an aneuploidy of a chromosome of interest in amaternal test sample comprising fetal and maternal nucleic acidmolecules by: (a) obtaining sequence information for the fetal andmaternal nucleic acids in the sample to identify a number of mappedsequence tags for a chromosome of interest and a number of sequence tagsfor at least two normalizing chromosomes; (b) using the number of tagsfor the chromosome of interest and the number of tags for a firstnormalizing chromosome to determine a first normalizing value for thechromosome of interest, and using the number of sequence tags for thefirst normalizing chromosome and the number of sequence tags for asecond normalizing chromosome to determine a second normalizing valuefor the first normalizing chromosome; and (c) comparing the firstnormalizing value for the chromosome of interest to a first thresholdvalue and comparing the second normalizing value for the firstnormalizing chromosome to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. The first andsecond threshold values can be the same or they can be different. Instep (c) of this method, the comparison of the first normalizing valuefor said chromosome of interest to a threshold value indicates thepresence or absence of an aneuploidy for said chromosome of interest,and the comparison of the second normalizing value for said firstnormalizing chromosome to a threshold value verifies the determinationof the presence or absence of an aneuploidy for the chromosome ofinterest. In some embodiments, the first normalizing value is a firstchromosome dose, which is a ratio of the number of sequence tags forsaid chromosome of interest and a first normalizing chromosome, and thesecond normalizing value a second chromosome dose, which is a ratio ofthe number of sequence tags for the first normalizing chromosome and asecond normalizing chromosome. Optionally, the first and secondnormalizing values can be expressed as normalized chromosome values(NCV) calculated as described below.

In another embodiment, the method verifies the determination of thepresence or absence of an aneuploidy of a chromosome of interest in amaternal test sample comprising fetal and maternal nucleic acidmolecules by: (a) obtaining sequence information for the fetal andmaternal nucleic acids in the sample to identify a number of mappedsequence tags for a chromosome of interest and a number of sequence tagsfor at least two normalizing chromosomes; (b) using the number of tagsfor the chromosome of interest and the number of tags for a firstnormalizing chromosome to determine a first normalizing value for thechromosome of interest, and using the number of sequence tags for thefirst normalizing chromosome and the number of sequence tags for asecond normalizing chromosome to determine a second normalizing valuefor the first normalizing chromosome; and (c) comparing the firstnormalizing value for the chromosome of interest to a first thresholdvalue and comparing the second normalizing value for the firstnormalizing chromosome to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. The first andsecond threshold values can be the same or they can be different. Instep (c) of this method, the comparison of the first normalizing valuefor said chromosome of interest to a threshold value indicates thepresence or absence of an aneuploidy for said chromosome of interest,and the comparison of the second normalizing value for said firstnormalizing chromosome to a threshold value verifies the determinationof the presence or absence of an aneuploidy for the chromosome ofinterest. In some embodiments, the first normalizing value is a firstchromosome dose, which is a ratio of the number of sequence tags forsaid chromosome of interest and a first normalizing chromosome, and thesecond normalizing value a second chromosome dose, which is a ratio ofthe number of sequence tags for the first normalizing chromosome and asecond normalizing chromosome. Optionally, the first and secondnormalizing values can be expressed as normalized chromosome values(NCV) calculated as described below. The fetal chromosomal aneuploidycan be a partial or a complete chromosomal aneuploidy. In theseembodiments, the fetal chromosomal aneuploidy can be selected from T21,T13, T18, and Monosomy X. In some embodiments, the maternal sample isobtained from a pregnant woman. In some embodiments, the maternal sampleis a biological fluid sample e.g. a blood sample or the plasma fractionderived therefrom. In some embodiments, the maternal sample is a plasmasample. In some embodiments, the nucleic acids in the maternal sampleare ctDNA molecules. In some other embodiments, the maternal test sampleis a plasma sample obtained from a pregnant woman and the nucleic acidmolecules are cfDNA molecules.

In another embodiment, the method determines the presence or absence ofat least two different fetal chromosomal aneuploidies in a maternal testsample comprising fetal and maternal nucleic acid molecules by repeatingthe steps (a)-(c) for at least two chromosomes of interest, whereinsteps (a)-(c) for each of the at least two chromosomes of interestcomprise (a) obtaining sequence information for the fetal and maternalnucleic acids in the sample to identify a number of mapped sequence tagsfor a chromosome of interest and a number of sequence tags for at leasttwo normalizing chromosomes; (b) using the number of tags for thechromosome of interest and the number of tags for a first normalizingchromosome to determine a first normalizing value for the chromosome ofinterest, and using the number of sequence tags for the firstnormalizing chromosome and the number of sequence tags for a secondnormalizing chromosome to determine a second normalizing value for thefirst normalizing chromosome; and (c) comparing the first normalizingvalue for the chromosome of interest to a first threshold value andcomparing the second normalizing value for the first normalizingchromosome to a second threshold value to determine the presence orabsence of a fetal aneuploidy in the sample. The first and secondthreshold values can be the same or they can be different. In step (c)of this method, for each of the at least two chromosomes of interest,the comparison of the first normalizing value for said chromosome ofinterest to a threshold value indicates the presence or absence of ananeuploidy for said chromosome of interest, and the comparison of thesecond normalizing value for said first normalizing chromosome to athreshold value verifies the determination of the presence or absence ofan aneuploidy for the chromosome of interest. In some embodiments, thefirst normalizing value is a first chromosome dose, which is a ratio ofthe number of sequence tags for said chromosome of interest and a firstnormalizing chromosome, and the second normalizing value a secondchromosome dose, which is a ratio of the number of sequence tags for thefirst normalizing chromosome and a second normalizing chromosome.Optionally, the first and second normalizing values can be expressed asnormalized chromosome values (NCV) as described herein. In someembodiments, the method comprises repeating the method for allchromosomes to determine the presence or absence of at least twodifferent fetal chromosomal aneuploidies.

In another embodiment, the method determines the presence or absence ofat least two different fetal chromosomal aneuploidies in a maternal testsample comprising fetal and maternal nucleic acid molecules by repeatingthe steps (a)-(c) for at least two chromosomes of interest, whereinsteps (a)-(c) for each of the at least two chromosomes of interestcomprise (a) obtaining sequence information for the fetal and maternalnucleic acids in the sample to identify a number of mapped sequence tagsfor a chromosome of interest and a number of sequence tags for at leasttwo normalizing chromosomes; (b) using the number of tags for thechromosome of interest and the number of tags for a first normalizingchromosome to determine a first normalizing value for the chromosome ofinterest, and using the number of sequence tags for the firstnormalizing chromosome and the number of sequence tags for a secondnormalizing chromosome to determine a second normalizing value for thefirst normalizing chromosome; and (c) comparing the first normalizingvalue for the chromosome of interest to a first threshold value andcomparing the second normalizing value for the first normalizingchromosome to a second threshold value to determine the presence orabsence of a fetal aneuploidy in the sample. The first and secondthreshold values can be the same or they can be different. In step (c)of this method, for each of the at least two chromosomes of interest,the comparison of the first normalizing value for said chromosome ofinterest to a threshold value indicates the presence or absence of ananeuploidy for said chromosome of interest, and the comparison of thesecond normalizing value for said first normalizing chromosome to athreshold value verifies the determination of the presence or absence ofan aneuploidy for the chromosome of interest. In some embodiments, thefirst normalizing value is a first chromosome dose, which is a ratio ofthe number of sequence tags for said chromosome of interest and a firstnormalizing chromosome, and the second normalizing value a secondchromosome dose, which is a ratio of the number of sequence tags for thefirst normalizing chromosome and a second normalizing chromosome.Optionally, the first and second normalizing values can be expressed asnormalized chromosome values (NCV) as described herein. In someembodiments, the method comprises repeating the method for allchromosomes to determine the presence or absence of at least twodifferent fetal chromosomal aneuploidies. The at least two differentfetal chromosomal aneuploidies can be selected from T21, T18, T13, andmonosomy X. In some embodiments, the maternal sample is obtained from apregnant woman. In some embodiments, the maternal sample is a biologicalfluid sample e.g. a blood sample or the plasma fraction derivedtherefrom. In some embodiments, the maternal sample is a plasma sample.In some embodiments, the nucleic acids in the maternal sample are cfDNAmolecules. In some other embodiments, the maternal test sample is aplasma sample obtained from a pregnant woman and the nucleic acidmolecules are cfDNA molecules.

In another embodiment, the method determines the presence or absence ofa fetal chromosomal aneuploidy selected from trisomy 21, trisomy 18,trisomy 13, and monosomy X, in a maternal plasma test sample comprisingfetal and maternal nucleic acid molecules e.g. cfDNA, by: (a) obtainingsequence information for the fetal and maternal nucleic acids in thematernal sample to identify a number of sequence tags for a chromosomeof interest and a number of sequence tags for at least two normalizingchromosomes, wherein obtaining the sequence information comprisesmassively parallel sequencing-by-synthesis using reversible dyeterminators; (b) using the number of sequence tags to calculate a firstand a second normalizing value for the chromosome of interest; and (c)comparing the first normalizing value for the chromosome of interest toa first threshold value and comparing the second normalizing value forthe chromosome of interest to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. In someembodiments, the first normalizing value is a first chromosome dose,which is a ratio of the number of sequence tags for the chromosome ofinterest and a first normalizing chromosome, and the second normalizingvalue is a second chromosome dose, which is a ratio of the number ofsequence tags for the chromosome of interest and a second normalizingchromosome. Optionally, the first and second normalizing values can beexpressed as normalized chromosome values 3.0 (NCV) as described herein.In some embodiments, the method determines the presence or absence of atleast two different chromosomal aneuploidies selected from trisomy 21,trisomy 18, trisomy 13, and monosomy X, in a maternal plasma test samplecomprising fetal and maternal nucleic acid molecules e.g. cfDNA, byrepeating steps (a)-(c) for at least two chromosomes of interest. Themethod can further comprise repeating the steps (a)-(c) for allchromosomes to determine the presence or absence of at least two fetalchromosomal aneuploidies. In some embodiments, the maternal sample isobtained from a pregnant woman. In some embodiments, the maternal sampleis a biological fluid sample e.g. a blood sample or the plasma fractionderived therefrom. In some embodiments, the maternal sample is a plasmasample. In some embodiments, the nucleic acids in the maternal sampleare cfDNA molecules. In some other embodiments, the maternal test sampleis a plasma sample obtained from a pregnant woman and the nucleic acidmolecules are cfDNA molecules.

In another embodiment, the method determines the presence or absence ofa fetal chromosomal aneuploidy selected from trisomy 21, trisomy 18,trisomy 13, and monosomy X, in a maternal plasma test sample comprisingfetal and maternal nucleic acid molecules e.g. cfDNA, by: (a) obtainingsequence information for the fetal and maternal nucleic acids in thesample to identify a number of mapped sequence tags for a chromosome ofinterest and a number of sequence tags for at least two normalizingchromosomes, wherein obtaining the sequence information comprisesmassively parallel sequencing-by-synthesis using reversible dyeterminators; (b) using the number of tags for the chromosome of interestand the number of tags for a first normalizing chromosome to determine afirst normalizing value for the chromosome of interest, and using thenumber of sequence tags for the first normalizing chromosome and thenumber of sequence tags for a second normalizing chromosome to determinea second normalizing value for the first normalizing chromosome; and (c)comparing the first normalizing value for the chromosome of interest toa first threshold value and comparing the second normalizing value forthe first normalizing chromosome to a second threshold value todetermine the presence or absence of a fetal aneuploidy in the sample.In some embodiments, the first normalizing value is a first chromosomedose, which is a ratio of the number of sequence tags for saidchromosome of interest and a first normalizing chromosome, and thesecond normalizing value a second chromosome dose, which is a ratio ofthe number of sequence tags for the first normalizing chromosome and asecond normalizing chromosome. Optionally, the first and secondnormalizing values can be expressed as normalized chromosome values(NCV) as described herein. In some embodiments, the method determinesthe presence or absence of at least two different chromosomalaneuploidies selected from trisomy 21, trisomy 18, trisomy 13, andmonosomy X, in a maternal plasma test sample comprising fetal andmaternal nucleic acid molecules e.g. cfDNA, by repeating steps (a)-(c)for at least two chromosomes of interest. The method can furthercomprise repeating the steps (a)-(c) for all chromosomes to determinethe presence or absence of at least two fetal chromosomal aneuploidies.In some embodiments, the maternal sample is obtained from a pregnantwoman. In some embodiments, the maternal sample is a biological fluidsample e.g. a blood sample or the plasma fraction derived therefrom. Insome embodiments, the maternal sample is a plasma sample. In someembodiments, the nucleic acids in the maternal sample are cfDNAmolecules. In some other embodiments, the maternal test sample is aplasma sample obtained from a pregnant woman and the nucleic acidmolecules are cfDNA molecules.

In some of the above and some of the subsequent embodiments, obtainingsequence information for the fetal and maternal nucleic acids in thesample comprises sequencing fetal and maternal nucleic acid molecules inthe sample.

INCORPORATION BY REFERENCE

All patents, patent applications, and other publications, including allsequences disclosed within these references, referred to herein areexpressly incorporated by reference, to the same extent as if eachindividual publication, patent or patent application was specificallyand individually indicated to be incorporated by reference. The citationof any document is not to be construed as an admission that it is priorart with respect to the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 provides a flowchart showing two alternate embodiments of themethod that determines and verifies the presence or absence of ananeuploidy.

FIG. 2 shows normalized chromosome values for chromosomes 21 (◯), 18(Δ), and 13 (□) determined in samples from training set 1 (Example 1).

FIG. 3 shows normalized chromosome values for chromosomes 21 (◯), 18(Δ), and 13 (□) determined in samples from test set 1 (Example 1).

FIG. 4 shows normalized chromosome values for chromosomes 21 (◯) and 18(Δ) determined in samples from test set 1 using the normalizing methodof Chiu et al. (Example 1).

FIG. 5 shows a plot of Normalized Chromosome Values for doses ofchromosome 9 determined in 48 samples in Test set 1 (Example 1) usingchromosome 11 as the normalizing chromosome.

FIG. 6 shows a plot of Normalized Chromosome Values for doses ofchromosome 8 determined in 48 samples in Test set 1 (Example 1) usingchromosome 2 as the normalizing chromosome.

FIG. 7 shows a plot of Normalized Chromosome Values for doses ofchromosome 6 determined in 48 samples in Test set 1 (Example 1) usingchromosome 5 as the normalizing chromosome.

FIG. 8 shows a plot of Normalized Chromosome Values for doses ofchromosome 21 determined in 48 samples in Test set 1 comprisingunaffected (◯) and affected (Δ) i.e. trisomy 21 samples, usingchromosome 9 (A), chromosome 10 (B), and chromosome 14 (C),respectively.

FIG. 9 shows a plot of Normalized Chromosome Values for doses ofchromosome 8 determined in Test Set 2 (Example 4) using chromosome 2 asthe normalizing chromosome (A), and using chromosome 7 as thenormalizing chromosome (B).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method capable of determining single ormultiple fetal chromosomal aneuploidies in a maternal sample comprisingfetal and maternal nucleic acids, and verifying that the correctdetermination has been made. The method is applicable to determiningcopy number variations (CNV) of any sequence of interest in samplescomprising mixtures of genomic nucleic acids derived from two differentgenomes, and which are known or are suspected to differ in the amount ofone or more sequence of interest. The method is applicable at least tothe practice of noninvasive prenatal diagnostics, and to the diagnosisand monitoring of conditions associated with a difference in sequencerepresentation in healthy versus diseased individuals.

Unless otherwise indicated, the practice of the present inventioninvolves conventional techniques commonly used in molecular biology,microbiology, protein purification, protein engineering, protein and DNAsequencing, and recombinant DNA fields, which are within the skill ofthe art. Such techniques are known to those of skill in the art and aredescribed in numerous texts and reference works (See e.g., Sambrook etal., “Molecular Cloning: A Laboratory Manual”, Second Edition (ColdSpring Harbor), [1989]); and Ausubel et al., “Current Protocols inMolecular Biology” [1987]).

Numeric ranges are inclusive of the numbers defining the range. It isintended that every maximum numerical limitation given throughout thisspecification includes every lower numerical limitation, as if suchlower numerical limitations were expressly written herein. Every minimumnumerical limitation given throughout this specification will includeevery higher numerical limitation, as if such higher numericallimitations were expressly written herein. Every numerical range giventhroughout this specification will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to theSpecification as a whole. Accordingly, as indicated above, the termsdefined immediately below are more fully defined by reference to thespecification as a whole.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the present invention, some preferred methods andmaterials are described. Accordingly, the terms defined immediatelybelow are more fully described by reference to the Specification as awhole. It is to be understood that this invention is not limited to theparticular methodology, protocols, and reagents described, as these mayvary, depending upon the context they are used by those of skill in theart.

DEFINITIONS

As used herein, the singular terms “a”, “an,” and “the” include theplural reference unless the context clearly indicates otherwise. Unlessotherwise indicated, nucleic acids are written left to right in 5′ to 3′orientation and amino acid sequences are written left to right in aminoto carboxy orientation.

The term “obtaining sequence information” herein refers to sequencingnucleic acids to obtain sequence information in the form of sequencereads, which when uniquely mapped to a reference genome are identifiedas sequence tags.

The term “normalizing value” herein refers to a numerical value that isdetermined for a chromosome of interest and that relates the number ofsequence tags for the chromosome of interest to the number of sequencetags for a normalizing chromosome. For example, a “normalizing value”can be a chromosome dose as described elsewhere herein, or it can be anNCV (Normalized Chromosome Value) as described elsewhere herein.

The term “chromosome of interest” herein refers to a chromosome forwhich a determination of the presence or absence of an aneuploidy ismade. Examples of chromosomes of interest include chromosomes that areinvolved in common aneuploidies such as trisomy 21, and chromosomes thatare involved in rare aneuploidies such as trisomy 2. Any one ofchromosomes 1-22, X and Y can be chromosomes of interest.

The terms “multiple” and “plurality” when used in reference to a numberof chromosomal aneuploidies and/or a number of chromosomes, hereinrefers to two or more aneuploidies and/or chromosomes.

The term “threshold value” herein refers to any number that iscalculated using a training data set and serves as a limit of diagnosisof a copy number variation e.g. an aneuploidy, in an organism. If athreshold is exceeded by results obtained from practicing the invention,a subject can be diagnosed with a copy number variation e.g. trisomy 21.Appropriate threshold values for the methods described herein can beidentified by analyzing normalizing values e.g. chromosome doses, orNCVs (normalized chromosome values) calculated for a training set ofsamples comprising qualified samples i.e. unaffected samples. Thresholdvalues can be set using qualified samples and samples identified ashaving chromosomal aneuploidies i.e. affected samples (see the Examplesherein). In some embodiments, the training set used to identifyappropriate threshold values comprises at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, at least 100, at least 200, at least 300, at least 400,at least 500, at least 600, at least 700, at least 800, at least 900, atleast 1000, at least 2000, at least 3000, at least 4000, or morequalified samples. It may advantageous to use larger sets of qualifiedsamples to improve the diagnostic utility of the threshold values.

The term “Next Generation Sequencing (NGS)” herein refers to sequencingmethods that allow for massively parallel sequencing of clonallyamplified and of single nucleic acid molecules. Non-limiting examples ofNGS include sequencing-by-synthesis using reversible dye terminators,and sequencing-by-ligation.

The term “read” refers to a DNA sequence of sufficient length (e.g., atleast about 30 bp) that can be used to identify a larger sequence orregion, e.g. that can be aligned and specifically assigned to achromosome or genomic region or gene.

The term “sequence tag” is herein used interchangeably with the term“mapped sequence tag” to refer to a sequence read that has beenspecifically assigned i.e. mapped, to a larger sequence e.g. a referencegenome, by alignment. Mapped sequence tags are uniquely mapped to areference genome i.e. they are assigned to a single location to thereference genome. Tags that can be mapped to more than one location on areference genome i.e. tags that do not map uniquely, are not included inthe analysis.

The term “number of sequence tags” when used in reference to the numberof tags for a chromosome of interest and/or normalizing chromosome(s)herein refers to the sequence tags that map to the chromosome ofinterest and/or normalizing chromosome(s) that are a subset of theplurality of tags obtained for all chromosomes in the sample. The numberof tags obtained for a sample can be at least about 1×10⁶ sequence tags,at least about 2×10⁶ sequence tags, at least about 3×10⁶ sequence tags,at least about 5×10⁶ sequence tags, at least about 8×10⁶ sequence tags,at least about 10×10⁶ sequence tags, at least about 15×10⁶ sequencetags, at least about 20×10⁶ sequence tags, at least about 30×10⁶sequence tags, at least about 40×10⁶ sequence tags, or at least about50×10⁶ sequence tags, or at least about 60×10⁶ sequence tags, or atleast about 70×10⁶ sequence tags, or at least about 80×10⁶ sequencetags, comprising between 20 and 40 bp reads e.g. 36 bp, are obtainedfrom mapping the reads to the reference genome per sample. The number oftags mapped to any one chromosome will depend on the size of thechromosome and the copy number of the chromosome. For example, thenumber of tags that map to chromosome 21 in a trisomy 21 sample will bedifferent i.e. greater than the number of tags mapped to a chromosome 21in an unaffected sample. Similarly, the number of tags mapped tochromosome 19 will be less than the number of tags that map tochromosome 1, which is about four times the size of chromosome 19. Thenumber of tags mapped to a sequence of interest e.g. a chromosome, isalso known as “sequence tag density”.

The term “sequence tag density” herein refers to the number of sequencereads that are mapped to a reference genome sequence e.g. the sequencetag density for chromosome 21 is the number of sequence reads generatedby the sequencing method that are mapped to chromosome 21 of thereference genome. Sequence tag density can be determined for wholechromosomes, or for portions of chromosomes.

As used herein, the terms “aligned”, “alignment”, or “aligning” refer toone or more sequences that are identified as a match in terms of theorder of their nucleic acid molecules to a known sequence from areference genome. Such alignment can be done manually or by a computeralgorithm, examples including the Efficient Local Alignment ofNucleotide Data (ELAND) computer program distributed as part of theIllumina Genomics Analysis pipeline. The matching of a sequence read inaligning can be a 100% sequence match or less than 100% (non-perfectmatch).

As used herein, the term “reference genome” refers to any particularknown genome sequence, whether partial or complete, of any organism orvirus which may be used to reference identified sequences from asubject. For example, a reference genome used for human subjects as wellas many other organisms is found on the world wide web at the NationalCenter for Biotechnology Information.

A “genome” refers to the complete genetic information of an organism orvirus, expressed in nucleic acid sequences.

The term “maternal sample” herein refers to a biological sample obtainedfrom a pregnant subject e.g. a woman.

The term “biological fluid” herein refers to a liquid taken from abiological source and includes, for example, blood, serum, plasma,sputum, lavage fluid, cerebrospinal fluid, urine, semen, sweat, tears,saliva, and the like. As used herein, the terms “blood,” “plasma” and“serum” expressly encompass fractions or processed portions thereof.Similarly, where a sample is taken from a biopsy, swab, smear, etc., the“sample” expressly encompasses a processed fraction or portion derivedfrom the biopsy, swab, smear, etc.

The terms “maternal nucleic acids” and “fetal nucleic acids” hereinrefer to the nucleic acids of a pregnant female subject and the nucleicacids of the fetus being carried by the pregnant female, respectively.

The term “subject” herein refers to a human subject as well as anon-human subject such as a mammal, an invertebrate, a vertebrate, afungus, a yeast, a bacteria, and a virus. Although the examples hereinconcern humans and the language is primarily directed to human concerns,the concept of this invention is applicable to genomes from any plant oranimal, and is useful in the fields of veterinary medicine, animalsciences, and research laboratories and such.

The term “normalizing sequence” herein refers to a sequence thatdisplays a variability in the number of sequence tags that are mapped toit among samples and sequencing runs that best approximates that of thesequence of interest for which it is used as a normalizing parameter,and that can best differentiate an affected sample from one or moreunaffected samples. A “normalizing chromosome” is an example of a“normalizing sequence”.

The term “sequence dose” herein refers to a parameter that relates thesequence tag density of a sequence of interest to the tag density of anormalizing sequence. A “chromosome dose”, which is a ratio of thenumber of sequence tags mapped to a chromosome e.g. a chromosome ofinterest, and the number of sequence tags mapped to a normalizingchromosome is an example of a sequence dose. A “test sequence dose” is aparameter that relates the sequence tag density of a sequence ofinterest e.g. chromosome 21, to that of a normalizing sequence e.g.chromosome 9, determined in a test sample. Similarly, a “qualifiedsequence dose” is a parameter that relates the sequence tag density of asequence of interest to that of a normalizing sequence determined in aqualified sample.

The term “chromosome dose” herein refers to a ratio of the number ofsequence tags mapped to a chromosome e.g. a chromosome of interest, andthe number of sequence tags mapped to a normalizing chromosome.

The term “normalizing chromosome” herein refers to a chromosome thatdisplays a variability in the number of sequence tags that are mapped toit among samples and sequencing runs that best approximates that of thechromosome of interest for which it is used to obtain a normalizingvalue, and that can best differentiate an affected sample from one ormore unaffected samples.

The term “sequence of interest” herein refers to a nucleic acid sequencethat is associated with a difference in sequence representation inhealthy versus diseased individuals. A sequence of interest can be asequence on a chromosome that is misrepresented i.e. over- orunder-represented, in a disease or genetic condition. A sequence ofinterest may also be a portion of a chromosome, or a chromosome i.e.chromosome of interest. For example, a sequence of interest can be achromosome that is over-represented in an aneuploidy condition e.g.chromosomes 13, 18, 21, and X, or a gene encoding a tumor-suppressorthat is under-represented in a cancer. Sequences of interest includesequences that are over- or under-represented in the total population,or a subpopulation of cells of a subject. A “qualified sequence ofinterest” is a sequence of interest in a qualified sample. A “testsequence of interest” is a sequence of interest in a test sample.

The term “qualified sample” herein refers to a sample comprising amixture of nucleic acids that are present in a known copy number towhich the nucleic acids in a test sample are compared, and it is asample that is normal i.e. not aneuploid, for the sequence of intereste.g. a qualified sample used for identifying a normalizing chromosomefor chromosome 21 is a sample that is not a trisomy 21 sample.

The terms “training set” and “training samples” are used herein to referto samples comprising nucleic acids that are present in a known copynumber to which the nucleic acids in a test sample are compared. Unlessotherwise specified, a training set comprises qualified and affectedsamples.

The term “test sample” herein refers to a sample comprising a mixture ofnucleic acids comprising at least one nucleic acid sequence whose copynumber is suspected of having undergone variation. Nucleic acids presentin a test sample are referred to as “test nucleic acids”.

The term “aneuploidy” herein refers to an imbalance of genetic materialcaused by a loss or gain of a whole chromosome, or part of a chromosome.

The term “chromosomal aneuploidy” herein refers to an imbalance ofgenetic material caused by a loss or gain of a whole chromosome, andincludes germline aneuploidy and mosaic aneuploidy.

The terms “partial aneuploidy” and “partial chromosomal aneuploidy”herein refer to an imbalance of genetic material caused by a loss orgain of part of a chromosome e.g. partial monosomy and partial trisomy,and encompasses imbalances resulting from translocations, deletions andinsertions.

The terms “nucleic acid molecules”, “polynucleotide”, and “nucleicacids” are used interchangeably and refer to a covalently linkedsequence of nucleotides (i.e., ribonucleotides for RNA anddeoxyribonucleotides for DNA) in which the 3′ position of the pentose ofone nucleotide is joined by a phosphodiester group to the 5′ position ofthe pentose of the next, include sequences of any form of nucleic acid,including, but not limited to RNA, DNA and cfDNA molecules. The term“polynucleotide” includes, without limitation, single- anddouble-stranded polynucleotide.

The term “copy number variation (CNV)” herein refers to variation in thenumber of copies of a nucleic acid sequence that is present in a testsample in comparison with the copy number of the nucleic acid sequencepresent in a qualified sample i.e. normal sample. Copy number variationsinclude deletions, including microdeletions, insertions, includingmicroinsertions, duplications, multiplications, inversions,translocations and complex multi-site variants. CNV encompass completechromosomal aneuploidies and partial aneuplodies.

DESCRIPTION

The present invention provides a method capable of determining single ormultiple fetal chromosomal aneuploidies in a maternal sample comprisingfetal and maternal nucleic acids, and verifying that the correctdetermination has been made. The method is applicable to determiningcopy number variations (CNV) of any sequence of interest in samplescomprising mixtures of genomic nucleic acids derived from at least twodifferent genomes and which are known or are suspected to differ in theamount of one or more sequence of interest. Sequences of interestinclude genomic sequences ranging from hundreds of bases to tens ofmegabases to entire chromosomes that are known or are suspected to beassociated with a genetic or a disease condition. Examples of sequencesof interest include chromosomes associated with well known aneuploidiese.g. trisomy 21, and segments of chromosomes that are multiplied indiseases such as cancer e.g. partial trisomy 8 in acute myeloidleukemia.

The present method comprises obtaining sequencing information tocalculate chromosome doses for sequences of interest e.g. chromosomes,to determine the presence or absence of a single or multiple chromosomalaneuploidies in one or more maternal test samples, and comprisesverifying that the correct determination of the aneuploidy is made. Theaccuracy required for correctly determining whether a CNV e.g.aneuploidy, is present or absent in a sample, is predicated on thevariation of the number of sequence tags that map to the referencegenome among samples within a sequencing run (intra-run sequencingvariation), and the variation of the number of sequence tags that map tothe reference genome in different sequencing runs (inter-run sequencingvariation), which can obscure the effects of fetal chromosomalaneuploidies on the distribution of mapped sequence tags. For example,the variation can be particularly pronounced for tags that map toGC-rich or GC-poor reference sequences. To correct for such variation,the present method uses chromosome doses based on the knowledge ofnormalizing chromosomes (or groups of normalizing chromosomes), tointrinsically account for the accrued sequencing variability.

Normalizing Chromosomes and Chromosome Doses

Normalizing chromosomes are identified using sequence information from aset of qualified samples obtained from subjects known to comprise cellshaving a normal copy number for any one sequence of interest e.g.diploid for chromosome 21. The sequence information obtained from thequalified samples is also used for determining statistically meaningfulidentification of chromosomal aneuploidies in test samples (seeExamples). In one embodiment, the qualified samples are obtained frommothers pregnant with a fetus that has been confirmed using cytogeneticmeans to have a normal copy number of chromosomes e.g. diploid forchromosome 21. The biological qualified samples may be a biologicalfluid e.g. plasma, or any suitable sample as described below. In someembodiments, the qualified sample contains a mixture of nucleic acidmolecules e.g. cfDNA molecules. In some embodiments, the qualifiedsample is a maternal plasma sample that contains a mixture of fetal andmaternal cfDNA molecules.

Sequence information for normalizing chromosomes is obtained bysequencing at least a portion of the nucleic acids e.g. fetal andmaternal nucleic acids, using any known sequencing method. Preferably,any one of the Next Generation Sequencing (NGS) methods describedelsewhere herein is used to sequence the fetal and maternal nucleicacids as single or clonally amplified molecules. Millions of sequencereads of a predetermined length e.g. 36 bp, are generated by the NGStechnology, and are mapped to a reference genome to be counted assequence tags. At least a portion of the nucleic acids of each of thequalified samples is sequenced and the number of sequence tags mapped toeach chromosome is counted. In some embodiments, the number of sequencetags mapped to a chromosome can be normalized to the length of thequalified sequence of interest to which they are mapped. Sequence tagdensities that are determined as a ratio of the tag density relative tothe length of the sequence of interest are herein referred to as tagdensity ratios. Normalization to the length of the sequence of interestis not required, and may be included as a step to reduce the number ofdigits in a number to simplify it for human interpretation. As allqualified sequence tags are mapped and counted in each of the qualifiedsamples, the qualified sequence tag density for a sequence of intereste.g. a clinically-relevant sequence, in the qualified samples isdetermined, as are the sequence tag densities for additional sequencesfrom which normalizing sequences are identified subsequently.

Based on the calculated qualified tag densities, qualified sequencedoses e.g. a chromosome doses, for a sequence of interest e.g.chromosome 21, are determined each as the ratio of the sequence tagdensity for the sequence of interest and the qualified sequence tagdensity for additional sequences from which normalizing sequences areidentified subsequently. For example, chromosome doses for thechromosome of interest e.g. chromosome 21, are determined as a ratio ofthe sequence tag density of chromosome 21 and the sequence tag densityfor each of all the remaining chromosomes i.e. chromosomes 1-20,chromosome 22, chromosome X, and chromosome Y. Qualified sequence dosescan be determined for all chromosomes.

Subsequently, at least two normalizing sequences for a sequence ofinterest e.g. chromosome 21, are identified in the qualified samplesbased on the calculated sequence doses. For example, the qualifiednormalizing sequences for chromosome 21 are identified as the sequencesin qualified samples that have variation in sequence tag density thatbest approximate that of chromosome 21. For example, qualifiednormalizing sequences are sequences that have the smallest variability.In some embodiments, more than two normalizing sequences are identified.For example, normalizing chromosomes having the lowest variability foreach of all chromosomes 1-22, chromosome X, and chromosome Y aredetermined. Table 9 in Example 5 provides the four normalizingchromosomes that were determined to have the four lowest variabilitiesfor each of chromosomes 1-22, chromosome X, and chromosome Y.Variability can be represented numerically as a coefficient of variation(% CV) as is shown in the Examples. The normalizing sequences can alsobe sequences that best distinguish one or more qualified samples fromone or more affected samples i.e. the normalizing sequences aresequences that have the greatest differentiability. The level ofdifferentiability can be determined as a statistical difference betweenthe chromosome doses in a population of qualified samples and thechromosome dose(s) in one or more test samples. For example,differentiability can be represented numerically as a T-test value,which represents the statistical difference between the chromosome dosesin a population of qualified samples and the chromosome dose(s) in oneor more test samples. Alternatively, differentiability can berepresented numerically as a Normalized Chromosome Value (NCV), which isa z-score for chromosome doses as long as the distribution for the NCVis normal. In determining the z-score, the mean and standard deviationof chromosome doses in a set of qualified samples can be used.Alternatively, the mean and standard deviation of chromosome doses in atraining set comprising qualified samples and affected samples can beused. In other embodiments, the normalizing sequence is a sequence thathas the smallest variability and the greatest differentiability.

The method identifies sequences that inherently have similarcharacteristics and that are prone to similar variations among samplesand sequencing runs, and which are useful for determining sequence dosesin test samples.

Based on the identification of the normalizing sequence(s) in qualifiedsamples, one or more sequence doses e.g. chromosome doses, aredetermined for a sequence of interest e.g. chromosome 21, in a testsample using the sequence information that is obtained for the nucleicacids in the test sample. In some embodiments, at least two sequencedoses e.g. chromosome doses, are determined for a sequence of interest.For example, a first chromosome dose is determined for chromosome 21using chromosome 9 as a first normalizing chromosome, and a secondchromosome dose is determined for chromosome 21 using chromosome 11 asthe second normalizing chromosome. The test sequence doses can befurther expressed as NCVs, as described below. In some embodiments,classification of the test sample can be made by directly comparing thefirst test sequence dose for the chromosome of interest to a firstthreshold value and comparing the second test sequence dose to a secondthreshold value to determine the presence or absence of a chromosomalaneuploidy in the test sample. Comparison of two chromosome doses for achromosome of interest verifies the determination of the sampleclassification. Threshold values are chosen according to a user-definedthreshold of reliability to classify the sample as a “normal”, an“affected” or a “no call” sample. In other embodiments, a firstchromosome dose is determined for a chromosome of interest using a firstnormalizing chromosome, and a second chromosome dose is determined forthe first normalizing chromosome using a second normalizing chromosome.Classification of the test sample can be made by comparing the firstchromosome dose to a first threshold value and comparing the secondchromosome dose to a second threshold value to determine the presence orabsence of a chromosomal aneuploidy in the test sample. Comparison of achromosome dose for a chromosome of interest to a first thresholddetermines the presence or absence of aneuploidy for the chromosome ofinterest in the test sample, and comparison of the second chromosomedose for the normalizing chromosome to a second threshold verifies thedetermination of the sample classification. The test chromosome dosescan be further expressed as NCVs, as described below, where the firstand second chromosome doses are expressed as first and second NCVs; andclassification of test samples is made by comparing the first NCV to afirst threshold and the second NCV to a second threshold.

Although the examples herein concern complete chromosomal aneuploidies,the concept of this invention is applicable to partial aneuploidies. Inone embodiment, the sequence of interest is a segment of a chromosomeassociated with a partial aneuploidy, e.g. a chromosomal deletion orinsertion, or unbalanced chromosomal translocation, and the at least twonormalizing sequences are chromosomal segments that are not associatedwith the partial aneuploidy and whose variation in sequence tag densitybest approximates that of the chromosome segment associated with thepartial aneuploidy. Partial aneuploidies can be determined usingchromosome doses (see International Application PCT/US2010/058609 filedon Dec. 1, 2010, and U.S. patent application Ser. No. 12/958,352,entitled “Method for Determining Copy Number Variations”, which werefiled on Dec. 1, 2010, which are herein incorporated by reference intheir entirety). The presence or absence of a partial aneuploidy can beverified using at least two normalizing sequences according to thepresent method.

FIG. 1 provides a flow chart of two exemplary embodiments of the method100, which determines and verifies the presence or absence of achromosomal aneuploidy in a sample comprising a mixture of two genomese.g. a maternal sample.

In a first embodiment, the method determines the presence or absence ofa fetal chromosomal aneuploidy in a maternal test sample comprisingfetal and maternal nucleic acids by: (a) obtaining sequence informationfor the fetal and maternal nucleic acids in the maternal sample toidentify a number of sequence tags for a chromosome of interest and anumber of sequence tags for at least two normalizing chromosomes; (b)using the number of sequence tags to calculate a first and a secondnormalizing value for the chromosome of interest; and (c) comparing thefirst normalizing value for the chromosome of interest to a firstthreshold value and comparing the second normalizing value for thechromosome of interest to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. The first andsecond threshold values can be the same or they can be different. Instep (c) of this method, the comparison of the first normalizing valuefor said chromosome of interest to a threshold value indicates thepresence or absence of an aneuploidy for said chromosome of interest,and the comparison of the second normalizing value for said chromosomeof interest to a threshold value verifies the determination of thepresence or absence of an aneuploidy for the chromosome of interest. Insome embodiments, the first normalizing value is a first chromosomedose, which is a ratio of the number of sequence tags for the chromosomeof interest and a first normalizing chromosome, and the secondnormalizing value is a second chromosome dose, which is a ratio of thenumber of sequence tags for the chromosome of interest and a secondnormalizing chromosome. Optionally, the first and second normalizingvalues can be expressed as normalized chromosome values (NCV) asdescribed below.

The first embodiment is depicted according to steps 110, 120, 130, and140 of the method as shown in FIG. 1. Fetal and maternal nucleic acidsobtained from a maternal sample are sequenced to provide a number ofsequence tags (110). The sequence tags mapped to a chromosome ofinterest e.g. chromosome 21, and the sequence tags mapped to twonormalizing chromosomes e.g. chromosome 9 and chromosome 11, are countedand used to calculate a corresponding first and second normalizingvalues e.g. chromosome doses, for the chromosome of interest. In oneembodiment, at least two chromosome doses are the normalizing valuesthat are determined for each chromosome of interest. In one embodiment,the first normalizing value for the chromosome of interest is a firstchromosome dose, which is a ratio of the number of sequence tags for thechromosome of interest and a first normalizing chromosome, and thesecond normalizing value for the chromosome of interest is a secondchromosome dose, which is a ratio of the number of sequence tags for thechromosome of interest and a second normalizing chromosome (120). Thefirst normalizing value for the chromosome of interest i.e. firstchromosome dose, is compared to a first threshold value and the secondnormalizing value for the chromosome of interest i.e. second chromosomedose, is compared to a second threshold value (130), and thedetermination and verification of the presence or absence of achromosomal aneuploidy is made (140). Alternatively, the at least twochromosome doses are expressed as a first and second normalizedchromosome values (NCVs), which first NCV relates the first chromosomedose to the mean of the corresponding first chromosome dose in a set ofqualified samples, and the second NCV relates the second chromosome doseto the mean of the corresponding chromosome dose in the same set ofqualified samples as:

${NCV}_{ij} = \frac{x_{ij} - {\hat{\mu}}_{j}}{{\hat{\sigma}}_{j}}$

where {circumflex over (μ)}_(j) AND {circumflex over (σ)}_(j) are theestimated mean and standard deviation, respectively, for the j-thchromosome dose in a set of qualified samples, and x_(ij) is theobserved j-th chromosome dose for test sample i. The first and secondnormalizing values i.e. NCVs are each compared to a first and a secondthreshold, respectively (130), and the determination and verification ofthe presence or absence of a chromosomal aneuploidy is made (140). Themethod is capable of identifying very rare e.g. trisomy 9, and morecommon chromosomal aneuploidies, e.g. trisomy 21, and can identifymultiple chromosomal aneuploidies from sequencing information obtainedfrom a single sequencing run on a test sample nucleic acid e.g. cfDNA.As is shown in the Examples, sequence information obtained for a sampleto determine the presence or absence of trisomy 21, revealed that whilea trisomy 21 was absent, the sample contained a trisomy 9. In someembodiments, chromosomal aneuploidies are identified in any ofchromosomes 1-22, chromosome X and chromosome Y. The chromosomalaneuploidy can be identified in the chromosome of interest and/or in thefirst or second normalizing chromosome. In some embodiments, the methodidentifies multiple chromosomal aneuploidies selected from trisomy 21,trisomy 13, trisomy 18 and monosomy X.

In a second embodiment, the method verifies the determination of thepresence or absence of an aneuploidy of a chromosome of interest in amaternal test sample comprising fetal and maternal nucleic acidmolecules by: (a) obtaining sequence information for the fetal andmaternal nucleic acids in the sample to identify a number of mappedsequence tags for a chromosome of interest and a number of sequence tagsfor at least two normalizing chromosomes; (b) using the number of tagsfor the chromosome of interest and the number of tags for a firstnormalizing chromosome to determine a first normalizing value for thechromosome of interest, and using the number of sequence tags for thefirst normalizing chromosome and the number of sequence tags for asecond normalizing chromosome to determine a second normalizing valuefor the first normalizing chromosome; and (c) comparing the firstnormalizing value for the chromosome of interest to a first thresholdvalue and comparing the second normalizing value for the firstnormalizing chromosome to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. The first andsecond threshold values can be the same or they can be different. Instep (c) of this method, the comparison of the first normalizing valuefor said chromosome of interest to a threshold value indicates thepresence or absence of an aneuploidy for said chromosome of interest,and the comparison of the second normalizing value for said firstnormalizing chromosome to a threshold value verifies the determinationof the presence or absence of an aneuploidy for the chromosome ofinterest. In some embodiments, the first normalizing value is a firstchromosome dose, which is a ratio of the number of sequence tags forsaid chromosome of interest and a first normalizing chromosome, and thesecond normalizing value a second chromosome dose, which is a ratio ofthe number of sequence tags for the first normalizing chromosome and asecond normalizing chromosome. Optionally, the first and secondnormalizing values can be expressed as normalized chromosome values(NCV) calculated as

${NCV}_{ij} = \frac{x_{ij} - {\hat{\mu}}_{j}}{{\hat{\sigma}}_{j}}$as described above.

The second embodiment is depicted according to steps 110, 150, 160, and140 of the method as shown in FIG. 1. Fetal and maternal nucleic acidsobtained from a maternal sample are sequenced to provide a number ofsequence tags (110). The sequence tags mapped to a chromosome ofinterest e.g. chromosome 21, and the sequence tags mapped to anormalizing chromosome e.g. chromosome 9, are counted and used tocalculate a corresponding first normalizing value e.g. chromosome dose,for the chromosome of interest, and a second normalizing value e.g. achromosome dose, is calculated for the first normalizing chromosome as aratio of the sequence tags mapped to the first normalizing chromosomee.g. chromosome 9, and the number of sequence tags mapped to a secondnormalizing chromosome e.g. chromosome 11 (150). The first and secondnormalizing values i.e. chromosome doses are each compared to a firstand second threshold, respectively (160), and the determination andverification of the presence or absence of a chromosomal aneuploidy ismade (140). Alternatively, the two normalizing values i.e. the twochromosome doses, are expressed as a first and second normalizedchromosome values (NCVs), which first NCV relates the first chromosomedose to the mean of the corresponding first chromosome close in a set ofqualified samples, and the second NCV relates the second chromosome doseto the mean of the corresponding chromosome dose in the same set ofqualified samples as:

${NCV}_{ij} = \frac{x_{ij} - {\hat{\mu}}_{j}}{{\hat{\sigma}}_{j}}$where {circumflex over (μ)}_(j) AND {circumflex over (σ)}_(j) are theestimated mean and standard deviation, respectively, for the j-thchromosome dose in a set of qualified samples, and x_(ij) is theobserved j-th chromosome dose for test sample i. The first and secondnormalizing values i.e. NCVs are each compared to a predeterminedthreshold (160), and the determination and verification of the presenceor absence of a chromosomal aneuploidy is made (140).

As described previously, the method is capable of identifying rareaneuploidies e.g. trisomy 9, and common aneuploidies e.g. trisomy 21,chromosomal aneuploidies, and can identify multiple chromosomalaneuploidies from sequencing information obtained from a singlesequencing run on a test sample nucleic acid e.g. cfDNA. In someembodiments, single or multiple chromosomal aneuploidies are identifiedin any of chromosomes 1-22, chromosome X and chromosome Y. Thechromosomal aneuploidy can be identified in the chromosome of interestand/or in the first or second normalizing chromosome. In someembodiments, the method identifies single or multiple chromosomalaneuploidies selected from trisomy 21, trisomy 13, trisomy 18, trisomy9, and monosomy X.

Normalizing chromosomes can be determined in one or more separate setsof qualified samples. In some embodiments, normalizing chromosomes canbe determined in one or more sets of qualified samples for allchromosomes in a genome. Determining normalizing chromosomes for allchromosomes in a genome allows for the determination of chromosomalaneuploidies in each of the chromosomes of the genome using sequencinginformation obtained from a single sequencing run of nucleic acids froma test sample.

In all embodiments, normalizing chromosomes can be selected as follows.

Normalizing chromosomes for chromosome 1 are selected from chromosomes10, 11, 9 and 15. In one embodiment, the first and second normalizingchromosomes for chromosome 1 are chromosome 10 and chromosome 11.

Normalizing chromosomes for chromosome 2 are selected from chromosomes8, 7, 12, and 14. In one embodiment, the first and second chromosomenormalizing chromosomes for chromosome 2 are chromosome 8 and chromosome7.

Normalizing chromosomes for chromosome 3 are selected from chromosomes6, 5, 8, and 18. In one embodiment, the first and second chromosomenormalizing chromosomes for chromosome 3 are chromosome 6 and chromosome5.

Normalizing chromosomes for chromosome 4 are selected from 3, 5, 6, and13. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 4 are chromosome 13 and chromosome 5.

Normalizing chromosomes for chromosome 5 are selected from 6, 3, 8, and18. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 5 are chromosome 6 and chromosome 3.

Normalizing chromosomes for chromosome 6 are selected from 5, 3, 8, and18. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 6 are chromosome 5 and chromosome 3.

Normalizing chromosomes for chromosome 7 are selected from 12, 2, 14 and8. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 7 are chromosome 12 and chromosome 2.

Normalizing chromosomes for chromosome 8 are selected from 2, 7, 12, and3. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 8 are chromosome 2 and chromosome 3.

Normalizing chromosomes for chromosome 9 are selected from 11, 10, 1,and 14. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 9 are chromosome 11 and chromosome 10.

Normalizing chromosomes for chromosome 10 are selected from 1, 11, 9,and 15. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 10 are chromosome 1 and chromosome 11.

Normalizing chromosomes for chromosome 11 as the chromosome of interestare selected from 1, 10, 9, and 15. In one embodiment, the first andsecond chromosome normalizing chromosomes for chromosome 11 arechromosome 1 and chromosome 10.

Normalizing chromosomes for chromosome 12 are selected from 7, 14, 2,and 8. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 12 are chromosome 7 and chromosome 14.

Normalizing chromosomes for chromosome 13 are selected from chromosome4, the group of chromosomes 2-6, chromosome 5, and chromosome 6. In oneembodiment, the first and second chromosome normalizing chromosomes forchromosome 13 are chromosome 4 and the group of chromosomes 2-6,respectively. The group of chromosomes 2-6 can be used as a first or asecond normalizing chromosome for chromosome of interest 13, and as anormalizing chromosome for a first normalizing chromosome that is usedfor chromosome 13. In some embodiments, verification of all chromosomesin the group can be performed. Two groups of chromosomes can be used asfirst and second normalizing chromosomes for chromosome 13, wherein thechromosomes of the first group are different from the chromosomes of thesecond group.

Normalizing chromosomes for chromosome 14 are selected from 12, 7, 2,and 9. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 14 are chromosome 12 and chromosome 7.

Normalizing chromosomes for chromosome 15 are selected from 1, 10, 11,and 9. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 2 are chromosome 1 and chromosome 10.

Normalizing chromosomes for chromosome 16 are selected from 20, 17, 15,and 1. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 16 are chromosome 20 and chromosome 17.

Normalizing chromosomes for chromosome 17 are selected from 16, 20, 19and 22. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 17 are chromosome 16 and chromosome 20.

Normalizing chromosomes for chromosome 18 are selected from 8, 3, 2, and6. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 18 are chromosome 8 and chromosome 3.

Normalizing chromosomes for chromosome 19 are selected from 22, 17, 16,and 20. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 19 are chromosome 22 and chromosome 17.

Normalizing chromosomes for chromosome 20 are selected from 16, 17, 15,and 1. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 20 are chromosome 16 and chromosome 17.

Normalizing chromosomes for chromosome 21 are selected from 9, 11, 14and 1. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 21 are chromosome 9 and chromosome 11.

Normalizing chromosomes for chromosome 22 are selected from 19, 17, 16,and 20. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome 22 are chromosome 19 and chromosome 17.

Normalizing chromosomes for chromosome X are selected from 6, 5, 13, and3. In one embodiment, the first and second chromosome normalizingchromosomes for chromosome X are chromosome 6 and chromosome 5.

Normalizing chromosomes for chromosome Y are selected from the group ofchromosomes 2-6, chromosome 3, chromosome 4, and chromosome 5. Inanother embodiment, the first and second chromosome normalizingchromosomes for chromosome Y are chromosome 3 and the group ofchromosomes 2-6, respectively. The group of chromosomes 2-6 can be usedas a first or second normalizing chromosome for chromosome Y, or as anormalizing chromosome for a first normalizing chromosome that is usedfor chromosome Y e.g. chromosome 3. In some embodiments, all chromosomesin the group of 2-6 are verified for the absence of aneuploidy. Twogroups of chromosomes can be used as first and second normalizingchromosomes for chromosome 13, wherein the chromosomes of the firstgroup are different from the chromosomes of the second group. Asexemplified for chromosomes 13 and Y, a normalizing chromosome can be achromosomes or a group of chromosomes.

In some embodiments, the methods may involve analysis of sequence tagsfor 3 or 4 normalizing chromosomes, in addition to the chromosome ofinterest.

Therefore, in some embodiments, the method determines the presence orabsence of a fetal chromosomal aneuploidy in a maternal test samplecomprising fetal and maternal nucleic acids by: (a) obtaining sequenceinformation for the fetal and maternal nucleic acids in the maternalsample to identify a number of sequence tags for a chromosome ofinterest and a number of sequence tags for three normalizingchromosomes; (b) using the number of sequence tags to calculate first,second and third normalizing values for the chromosome of interest; and(c) comparing the first, second and third normalizing values for thechromosome of interest to one or more threshold values to determine thepresence or absence of a fetal aneuploidy in the maternal sample. Insome embodiments, the first normalizing value for the chromosome ofinterest is a first chromosome dose, which is a ratio of the number ofsequence tags for the chromosome of interest and a first normalizingchromosome, and the second normalizing value for the chromosome ofinterest is a second chromosome dose, which is a ratio of the number ofsequence tags for the chromosome of interest and a second normalizingchromosome, and the third normalizing value for the chromosome ofinterest is a third chromosome dose, which is a ratio of the number ofsequence tags for the chromosome of interest and a third normalizingchromosome. Optionally, the first, second and third normalizing valuescan be expressed as normalized chromosome values (NCV) as describedelsewhere herein.

Furthermore, in some embodiments, the method verifies the determinationof the presence or absence of an aneuploidy of a chromosome of interestin a maternal test sample comprising fetal and maternal nucleic acidmolecules by: (a) obtaining sequence information for the fetal andmaternal nucleic acids in the maternal sample to identify a number ofsequence tags for a chromosome of interest and a number of sequence tagsfor three normalizing chromosomes; (b) using the number of mapped tagsfor the chromosome of interest and the number of tags for a firstnormalizing chromosome to determine a first normalizing value for thechromosome of interest, (c) using the number of tags for the firstnormalizing chromosome and the number of tags for a second normalizingchromosome to determine a second normalizing value for the firstnormalizing chromosome; (d) using the number of tags for the secondnormalizing chromosome and the number of tags for a third normalizingchromosome to determine a third normalizing value for the secondnormalizing chromosome, and (e) comparing the first, second and thirdnormalizing values for the chromosome of interest to one or morethreshold values to determine the presence or absence of a fetalaneuploidy in the maternal sample. In some embodiments, the firstnormalizing value is a first chromosome dose, which is a ratio of thenumber of sequence tags for said chromosome of interest and a firstnormalizing chromosome, and the second normalizing value is a secondchromosome dose, which is a ratio of the number of sequence tags for thefirst normalizing chromosome and a second normalizing chromosome, andthe third normalizing value is a third chromosome dose, which is a ratioof the number of sequence tags for the second normalizing chromosome anda third normalizing chromosome. Optionally, the first, second and thirdnormalizing values can be expressed as normalized chromosome values(NCV) as described elsewhere herein.

In some embodiments, the method determines the presence or absence of afetal chromosomal aneuploidy in a maternal test sample comprising fetaland maternal nucleic acids by: (a) obtaining sequence information forthe fetal and maternal nucleic acids in the maternal sample to identifya number of sequence tags for a chromosome of interest and a number ofsequence tags for four normalizing chromosomes; (b) using the number ofsequence tags to calculate first, second, third and fourth normalizingvalues for the chromosome of interest; and (c) comparing the first,second, third and fourth normalizing values for the chromosome ofinterest to one or more threshold values to determine the presence orabsence of a fetal aneuploidy in the maternal sample. In someembodiments, the first normalizing value for the chromosome of interestis a first chromosome dose, which is a ratio of the number of sequencetags for the chromosome of interest and a first normalizing chromosome,and the second normalizing value for the chromosome of interest is asecond chromosome dose, which is a ratio of the number of sequence tagsfor the chromosome of interest and a second normalizing chromosome, andthe third normalizing value for the chromosome of interest is a thirdchromosome dose, which is a ratio of the number of sequence tags for thechromosome of interest and a third normalizing chromosome, and thefourth normalizing value for the chromosome of interest is a fourthchromosome dose, which is a ratio of the number of sequence tags for thechromosome of interest and a fourth normalizing chromosome. Optionally,the first, second, third and fourth normalizing values can be expressedas normalized chromosome values (NCV) as described elsewhere herein.

In some embodiments, the method determines and verifies the presence orabsence of an aneuploidy of a chromosome of interest in a maternal testsample comprising fetal and maternal nucleic acid molecules by: (a)obtaining sequence information for the fetal and maternal nucleic acidsin the maternal sample to identify a number of sequence tags for achromosome of interest and a number of sequence tags for fournormalizing chromosomes; (b) using the number of mapped tags for thechromosome of interest and the number of tags for a first normalizingchromosome to determine a first normalizing value for the chromosome ofinterest; (c) using the number of tags for the first normalizingchromosome and the number of tags for a second normalizing chromosome todetermine a second normalizing value for the first normalizingchromosome; and (d) using the number of tags for the second normalizingchromosome and the number of tags for a third normalizing chromosome todetermine a third normalizing value for the second normalizingchromosome; (e) using the number of tags for the third normalizingchromosome and the number of tags for a fourth normalizing chromosome todetermine a fourth normalizing value for the third normalizingchromosome; and (f) comparing the first, second, third, and four thenormalizing values for the chromosome of interest to one or morethreshold values to determine the presence or absence of a fetalaneuploidy in the maternal sample. In some embodiments, the firstnormalizing value is a first chromosome dose, which is a ratio of thenumber of sequence tags for said chromosome of interest and a firstnormalizing chromosome, and the second normalizing value is a secondchromosome dose, which is a ratio of the number of sequence tags for thefirst normalizing chromosome and a second normalizing chromosome, andthe third normalizing value is a third chromosome dose, which is a ratioof the number of sequence tags for the second normalizing chromosome anda third normalizing chromosome, and the fourth normalizing value is afourth chromosome dose, which is a ratio of the number of sequence tagsfor the third normalizing chromosome and a fourth normalizingchromosome. Optionally, the first, second, third and fourth normalizingvalues can be expressed as normalized chromosome values (NCV) asdescribed elsewhere herein.

In these embodiments the first, second, third and fourth normalizingchromosomes can be selected from those set out above. For example, thefirst, second, third and fourth normalizing chromosomes for chromosome 1can be selected from chromosomes 10, 11, 9 and 15; the first, second,third and fourth normalizing chromosomes for chromosome 2 can beselected from chromosomes 8, 7, 12, and 14; the first, second, third andfourth normalizing chromosomes for chromosome 3 can be selected fromchromosomes 6, 5, 8, and 18; the first, second, third and fourthnormalizing chromosomes for chromosome 4 can be selected fromchromosomes 3, 5, 6, and 13; the first, second, third and fourthnormalizing chromosomes for chromosome 5 can be selected fromchromosomes 6, 3, 8, and 18; the first, second, third and fourthnormalizing chromosomes for chromosome 6 can be selected fromchromosomes 5, 3, 8, and 18. the first, second, third and fourthnormalizing chromosomes for chromosome 7 can be selected fromchromosomes 12, 2, 14 and 8; the first, second, third and fourthnormalizing chromosomes for chromosome 8 can be selected fromchromosomes 2, 7, 12, and 3 the first, second, third and fourthnormalizing chromosomes for chromosome 9 can be selected fromchromosomes 11, 10, 1, and 14; the first, second, third and fourthnormalizing chromosomes for chromosome 10 can be selected from 1, 11, 9,and 15; the first, second, third and fourth normalizing chromosomes forchromosome 11 can be selected from chromosomes 1, 10, 9, and 15; thefirst, second, third and fourth normalizing chromosomes for chromosome12 can be selected from chromosomes 7, 14, 2, and 8; the first, second,third and fourth normalizing chromosomes for chromosome 13 can beselected from chromosomes 4, group of chromosomes 2-6, 5, and 6; thefirst, second, third and fourth normalizing chromosomes for chromosome14 can be selected from chromosomes 12, 7, 2, and 9; the first, second,third and fourth normalizing chromosomes for chromosome 15 can beselected from 1, 10, 11, and 9; the first, second, third and fourthnormalizing chromosomes for chromosome 16 can be selected fromchromosomes 20, 17, 15, and 1; the first, second, third and fourthnormalizing chromosomes for chromosome 17 can be selected fromchromosomes 16, 20, 19 and 22; the first, second, third and fourthnormalizing chromosomes for chromosome 18 can be selected fromchromosomes 8, 3, 2, and 6; the first, second, third and fourthnormalizing chromosomes for chromosome 19 can be selected fromchromosomes 22, 17, 16, and 20; the first, second, third and fourthnormalizing chromosomes for chromosome 20 can be selected fromchromosomes 16, 17, 15, and 1; the first, second, third and fourthnormalizing chromosomes for chromosome 21 can be selected fromchromosomes 9, 11, 14 and 1; the first, second, third and fourthnormalizing chromosomes for chromosome 22 can be selected fromchromosomes 19, 17, 16, and 20; the first, second, third and fourthnormalizing chromosomes for chromosome X can be selected fromchromosomes 6, 5, 13, and 3; and the first, second, third and fourthnormalizing chromosomes for chromosome Y can be selected from group ofchromosomes 2-6, chromosomes 3, 4, and 5.

Sequencing Methods

In some of the methods of the invention, obtaining sequence informationfor the fetal and maternal nucleic acids in the sample to identify anumber of sequence tags comprises sequencing fetal and maternal nucleicacid molecules in the sample.

Sequence information is obtained by sequencing genomic DNA e.g.cell-free DNA in a maternal sample, using any one of the Next GenerationSequencing (NGS) methods in which clonally amplified DNA templates orsingle DNA molecules, are sequenced in a massively parallel fashion(e.g. as described in Volkerding et al. Clin Chem 55:641-658 [2009];Metzker M Nature Rev 11:31-46 [2010]). In addition to high-throughputsequence information, NGS provides quantitative information, in thateach sequence read is a countable “sequence tag” representing anindividual clonal DNA template or a single DNA molecule. The sequencingtechnologies of NGS include without limitation pyrosequencing,sequencing-by-synthesis with reversible dye terminators, sequencing byoligonucleotide probe ligation and ion semiconductor sequencing. DNAfrom individual samples can be sequenced individually (i.e. singleplexsequencing) or DNA from multiple samples can be pooled and sequenced asindexed genomic molecules (i.e. multiplex sequencing) on a singlesequencing run, to generate up to several hundred million reads of DNAsequences. Examples of sequencing technologies that can be used toobtain the sequence information according to the present method aredescribed below.

Some of the sequencing technologies are available commercially, such asthe sequencing-by-hybridization platform from Affymetrix Inc.(Sunnyvale, Calif.) and the sequencing-by-synthesis platforms from 454Life Sciences (Bradford, Conn.), Illumina/Solexa (Hayward, Calif.) andHelicos Biosciences (Cambridge, Mass.), and the sequencing-by-ligationplatform from Applied Biosystems (Foster City, Calif.), as describedbelow. In addition to the single molecule sequencing performed usingsequencing-by-synthesis of Helicos Biosciences, other single moleculesequencing technologies include the SMRT™ technology of PacificBiosciences, the Ion Torrent™ technology, and nanopore sequencing beingdeveloped for example, by Oxford Nanopore Technologies. While theautomated Sanger method is considered as a ‘first generation’technology, the present method can be applied to bioassays that useSanger sequencing, including automated Sanger sequencing. In addition,the present method can be applied to bioassays that use nucleic acidimaging technologies e.g. atomic force microscopy (AFM) or transmissionelectron microscopy (TEM). Exemplary sequencing technologies aredescribed below.

In one embodiment, the present method comprises obtaining sequenceinformation for the genomic DNA e.g. fetal and maternal cfDNA, usingsingle molecule sequencing technology the Helicos True Single MoleculeSequencing (tSMS) technology (e.g. as described in Harris T. D. et al.,Science 320:106-109 [2008]). In the tSMS technique, a DNA sample iscleaved into strands of approximately 100 to 200 nucleotides, and apolyA sequence is added to the 3′ end of each DNA strand. Each strand islabeled by the addition of a fluorescently labeled adenosine nucleotide.The DNA strands are then hybridized to a flow cell, which containsmillions of oligo-T capture sites that are immobilized to the flow cellsurface. The templates can be at a density of about 100 milliontemplates/cm². The flow cell is then loaded into an instrument, e.g.,HeliScope™ sequencer, and a laser illuminates the surface of the flowcell, revealing the position of each template. A CCD camera can map theposition of the templates on the flow cell surface. The templatefluorescent label is then cleaved and washed away. The sequencingreaction begins by introducing a DNA polymerase and a fluorescentlylabeled nucleotide. The oligo-T nucleic acid serves as a primer. Thepolymerase incorporates the labeled nucleotides to the primer in atemplate directed manner. The polymerase and unincorporated nucleotidesare removed. The templates that have directed incorporation of thefluorescently labeled nucleotide are discerned by imaging the flow cellsurface. After imaging, a cleavage step removes the fluorescent label,and the process is repeated with other fluorescently labeled nucleotidesuntil the desired read length is achieved. Sequence information iscollected with each nucleotide addition step. Whole genome sequencing bysingle molecule sequencing technologies excludes PCR-based amplificationin the preparation of the sequencing libraries, and the directness ofsample preparation allows for direct measurement of the sample, ratherthan measurement of copies of that sample.

In another embodiment, the present method comprises obtaining sequenceinformation for the genomic DNA e.g. fetal and maternal cfDNA, using the454 sequencing (Roche) (e.g. as described in Margulies, M. et al. Nature437:376-380 [2005]). 454 sequencing involves two steps. In the firststep, DNA is sheared into fragments of approximately 300-800 base pairs,and the fragments are blunt-ended. Oligonucleotide adaptors are thenligated to the ends of the fragments. The adaptors serve as primers foramplification and sequencing of the fragments. The fragments can beattached to DNA capture beads, e.g., streptavidin-coated beads using,e.g., Adaptor B, which contains 5′-biotin tag. The fragments attached tothe beads are PCR amplified within droplets of an oil-water emulsion.The result is multiple copies of clonally amplified DNA fragments oneach bead. In the second step, the beads are captured in wells(pico-liter sized). Pyrosequencing is performed on each DNA fragment inparallel. Addition of one or more nucleotides generates a light signalthat is recorded by a CCD camera in a sequencing instrument. The signalstrength is proportional to the number of nucleotides incorporated.Pyrosequencing makes use of pyrophosphate (PPi) which is released uponnucleotide addition. PPi is converted to ATP by ATP sulfurylase in thepresence of adenosine 5′ phosphosulfate. Luciferase uses ATP to convertluciferin to oxyluciferin, and this reaction generates light that isdiscerned and analyzed.

In another embodiment, the present method comprises obtaining sequenceinformation for the genomic DNA e.g. fetal and maternal cfDNA, using theSOLiD™ technology (Applied Biosystems). In SOLiD™sequencing-by-ligation, genomic DNA is sheared into fragments, andadaptors are attached to the 5′ and 3′ ends of the fragments to generatea fragment library. Alternatively, internal adaptors can be introducedby ligating adaptors to the 5′ and 3′ ends of the fragments,circularizing the fragments, digesting the circularized fragment togenerate an internal adaptor, and attaching adaptors to the 5′ and 3′ends of the resulting fragments to generate a mate-paired library. Next,clonal bead populations are prepared in microreactors containing beads,primers, template, and PCR components. Following PCR, the templates aredenatured and beads are enriched to separate the beads with extendedtemplates. Templates on the selected beads are subjected to a 3′modification that permits bonding to a glass slide. The sequence can bedetermined by sequential hybridization and ligation of partially randomoligonucleotides with a central determined base (or pair of bases) thatis identified by a specific fluorophore. After a color is recorded, theligated oligonucleotide is cleaved and removed and the process is thenrepeated.

In another embodiment, the present method comprises obtaining sequenceinformation for the genomic DNA e.g. fetal and maternal cfDNA using thesingle molecule, real-time (SMRT™) sequencing technology of PacificBiosciences. In SMRT sequencing, the continuous incorporation ofdye-labeled nucleotides is imaged during DNA synthesis. Single DNApolymerase molecules are attached to the bottom surface of individualzero-mode wavelength identifiers (ZMW identifiers) that obtain sequenceinformation while phospholinked nucleotides are being incorporated intothe growing primer strand. A ZMW is a confinement structure whichenables observation of incorporation of a single nucleotide by DNApolymerase against the background of fluorescent nucleotides thatrapidly diffuse in an out of the ZMW (in microseconds). It takes severalmilliseconds to incorporate a nucleotide into a growing strand. Duringthis time, the fluorescent label is excited and produces a fluorescentsignal, and the fluorescent tag is cleaved off. Identification of thecorresponding fluorescence of the dye indicates which base wasincorporated. The process is repeated.

In another embodiment, the present method comprises obtaining sequenceinformation for the genomic DNA e.g. fetal and maternal cfDNA, usingnanopore sequencing (e.g. as described in Soni G V and Meller A. ClinChem 53: 1996-2001 [2007]). Nanopore sequencing DNA analysis techniquesare being industrially developed by a number of companies, includingOxford Nanopore Technologies (Oxford, United Kingdom). Nanoporesequencing is a single-molecule sequencing technology whereby a singlemolecule of DNA is sequenced directly as it passes through a nanopore. Ananopore is a small hole, of the order of 1 nanometer in diameter.Immersion of a nanopore in a conducting fluid and application of apotential (voltage) across it results in a slight electrical current dueto conduction of ions through the nanopore. The amount of current whichflows is sensitive to the size and shape of the nanopore. As a DNAmolecule passes through a nanopore, each nucleotide on the DNA moleculeobstructs the nanopore to a different degree, changing the magnitude ofthe current through the nanopore in different degrees. Thus, this changein the current as the DNA molecule passes through the nanoporerepresents a reading of the DNA sequence.

In another embodiment, the present method comprises obtaining sequenceinformation for the genomic DNA e.g. fetal and maternal cfDNA using thechemical-sensitive field effect transistor (chemFET) array (e.g., asdescribed in U.S. Patent Application Publication No. 20090026082). Inone example of the technique, DNA molecules can be placed into reactionchambers, and the template molecules can be hybridized to a sequencingprimer bound to a polymerase. Incorporation of one or more triphosphatesinto a new nucleic acid strand at the 3′ end of the sequencing primercan be discerned by a change in current by a chemFET. An array can havemultiple chemFET sensors. In another example, single nucleic acids canbe attached to beads, and the nucleic acids can be amplified on thebead, and the individual beads can be transferred to individual reactionchambers on a chemFET array, with each chamber having a chemFET sensor,and the nucleic acids can be sequenced.

In another embodiment, the present method comprises obtaining sequenceinformation for the genomic DNA e.g. fetal and maternal cfDNA using theHalcyon Molecular's technology, which uses transmission electronmicroscopy (TEM). The method, termed Individual Molecule Placement RapidNano Transfer (IMPRNT), comprises utilizing single atom resolutiontransmission electron microscope imaging of high-molecular weight (150kb or greater) DNA selectively labeled with heavy atom markers andarranging these molecules on ultra-thin films in ultra-dense (3 nmstrand-to-strand) parallel arrays with consistent base-to-base spacing.The electron microscope is used to image the molecules on the films todetermine the position of the heavy atom markers and to extract basesequence information from the DNA. The method is further described inPCT patent publication WO 2009/046445. The method allows for sequencingcomplete human genomes in less than ten minutes.

In another embodiment, the DNA sequencing technology is the Ion Torrentsingle molecule sequencing, which pairs semiconductor technology with asimple sequencing chemistry to directly translate chemically encodedinformation (A, C, G, T) into digital information (0, 1) on asemiconductor chip. In nature, when a nucleotide is incorporated into astrand of DNA by a polymerase, a hydrogen ion is released as abyproduct. Ion Torrent uses a high-density array of micro-machined wellsto perform this biochemical process in a massively parallel way. Eachwell holds a different DNA molecule. Beneath the wells is anion-sensitive layer and beneath that an ion sensor. When a nucleotide,for example a C, is added to a DNA template and is then incorporatedinto a strand of DNA, a hydrogen ion will be released. The charge fromthat ion will change the pH of the solution, which can be identified byIon Torrent's ion sensor. The sequencer—essentially the world's smallestsolid-state pH meter—calls the base, going directly from chemicalinformation to digital information. The Ion personal Genome Machine(PGM™) sequencer then sequentially floods the chip with one nucleotideafter another. If the next nucleotide that floods the chip is not amatch. No voltage change will be recorded and no base will be called. Ifthere are two identical bases on the DNA strand, the voltage will bedouble, and the chip will record two identical bases called. Directidentification allows recordation of nucleotide incorporation inseconds.

In another embodiment, the present method comprises obtaining sequenceinformation for the genomic DNA e.g. fetal and maternal cfDNA bymassively parallel sequencing of millions of DNA fragments usingIllumina's sequencing-by-synthesis and reversible terminator-basedsequencing chemistry (e.g. as described in Bentley et al., Nature6:53-59 [2009]). Template DNA can be genomic DNA e.g. cfDNA. In someembodiments, genomic DNA from isolated cells is used as the template,and it is fragmented into lengths of several hundred base pairs. Inother embodiments, cfDNA is used as the template, and fragmentation isnot required as cfDNA exists as short fragments. For example fetal cfDNAcirculates in the bloodstream as fragments of <300 bp, and maternalcfDNA has been estimated to circulate as fragments of between about 0.5and 1 Kb (Li et al., Clin Chem, 50: 1002-1011 [2004]). Illumina'ssequencing technology relies on the attachment of fragmented genomic DNAto a planar, optically transparent surface on which oligonucleotideanchors are bound. Template DNA is end-repaired to generate5′-phosphorylated blunt ends, and the polymerase activity of Klenowfragment is used to add a single A base to the 3′ end of the bluntphosphorylated DNA fragments. This addition prepares the DNA fragmentsfor ligation to oligonucleotide adapters, which have an overhang of asingle T base at their 3′ end to increase ligation efficiency. Theadapter oligonucleotides are complementary to the flow-cell anchors.Under limiting-dilution conditions, adapter-modified, single-strandedtemplate DNA is added to the flow cell and immobilized by hybridizationto the anchors. Attached DNA fragments are extended and bridge amplifiedto create an ultra-high density sequencing flow cell with hundreds ofmillions of clusters, each containing ˜1,000 copies of the sametemplate. In one embodiment, the randomly fragmented genomic DNA e.g.cfDNA, is amplified using PCR before it is subjected to clusteramplification. Alternatively, an amplification-free genomic librarypreparation is used, and the randomly fragmented genomic DNA e.g. cfDNAis enriched using the cluster amplification alone (Kozarewa et al.,Nature Methods 6:291-295 [2009]). The templates are sequenced using arobust four-color DNA sequencing-by-synthesis technology that employsreversible terminators with removable fluorescent dyes. High-sensitivityfluorescence identification is achieved using laser excitation and totalinternal reflection optics. Short sequence reads of about 20-40 bp e.g.36 bp, are aligned against a repeat-masked reference genome and geneticdifferences are called using specially developed data analysis pipelinesoftware. After completion of the first read, the templates can beregenerated in situ to enable a second read from the opposite end of thefragments. Thus, either single-end or paired end sequencing of the DNAfragments can be used. Partial sequencing of DNA fragments present inthe sample is performed, and sequence tags comprising reads ofpredetermined length e.g. 36 bp, are mapped to a known reference genome.The mapped tags can be counted.

In one embodiment, the reference genome sequence is the NCBI36/hg18sequence, which is available on the world wide web at the UCSC humangenome browser. In another embodiment, the reference genome sequence isthe GRCh37/hg19, which is available on the world wide web at the UCSChuman genome browser. The sequences of other reference genomes from avariety of species are available on the world wide web at the NCBIwebsite. Other sources of public sequence information include GenBank,dbEST, dbSTS, EMBL (the European Molecular Biology Laboratory), and theDDBJ (the DNA Databank of Japan). A number of computer algorithms areavailable for aligning sequences, including without limitation BLAST(Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993),FASTA (Person & Lipman, 1988), BOWTIE (Langmead et al., Genome Biology10:R25.1-R25.10 [2009]), or ELAND (Illumina, Inc., San Diego, Calif.,USA). In one embodiment, one end of the clonally expanded copies of theplasma cfDNA molecules is sequenced and processed by bioinformaticalignment analysis for the Illumina Genome Analyzer, which uses theEfficient Large-Scale Alignment of Nucleotide Databases (ELAND)software.

In some embodiments of the method described herein, the mapped sequencetags comprise sequence reads of about 20 bp, about 25 bp, about 30 bp,about 35 bp, about 40 bp, about 45 bp, about 50 bp, about 55 bp, about60 bp, about 65 bp, about 70 bp, about 75 bp, about 80 bp, about 85 bp,about 90 bp, about 95 bp, about 100 bp, about 110 bp, about 120 bp,about 130, about 140 bp, about 150 bp, about 200 bp, about 250 bp, about300 bp, about 350 bp, about 400 bp, about 450 bp, or about 500 bp. It isexpected that technological advances will enable single-end reads ofgreater than 500 bp enabling for reads of greater than about 1000 bpwhen paired end reads are generated. In one embodiment, the mappedsequence tags comprise sequence reads that are 36 bp. Mapping of thesequence tags is achieved by comparing the sequence of the tag with thesequence of the reference to determine the chromosomal origin of thesequenced nucleic acid (e.g. cfDNA) molecule, and specific geneticsequence information is not needed. A small degree of mismatch (0-2mismatches per sequence tag) may be allowed to account for minorpolymorphisms that may exist between the reference genome and thegenomes in the mixed sample.

A plurality of sequence tags are obtained per sample. In someembodiments, at least about 3×10⁶ sequence tags, at least about 5×10⁶sequence tags, at least about 8×10⁶ sequence tags, at least about 10×10⁶sequence tags, at least about 15×10⁶ sequence tags, at least about20×10⁶ sequence tags, at least about 30×10⁶ sequence tags, at leastabout 40×10⁶ sequence tags, or at least about 50×10⁶ sequence tagscomprising between 20 and 40 bp reads e.g. 36 bp, are obtained frommapping the reads to the reference genome per sample. In one embodiment,all the sequence reads are mapped to all regions of the referencegenome. In one embodiment, the tags that have been mapped to all regionse.g. all chromosomes, of the reference genome are counted, and the CNVi.e. the over- or under-representation of a sequence of interest e.g. achromosome or portion thereof, in the mixed DNA sample is determined.The method does not require differentiation between the two genomes.

In some embodiments, the method determines the presence or absence of afetal chromosomal aneuploidy in a maternal test sample comprising fetaland maternal nucleic acid molecules by (a) obtaining sequenceinformation for the fetal and maternal nucleic acids in the maternalsample to identify a number of sequence tags for a chromosome ofinterest and a number of sequence tags for at least two normalizingchromosomes, wherein the sequence information comprises next generationsequencing (NGS); comprises sequencing-by-synthesis using reversible dyeterminators; comprises sequencing-by-ligation; or comprises singlemolecule sequencing; (b) using the number of sequence tags to calculatea first and a second normalizing value for the chromosome of interest;and (c) comparing the first normalizing value for the chromosome ofinterest to a first threshold value and comparing the second normalizingvalue for the chromosome of interest to a second threshold value todetermine the presence or absence of a fetal aneuploidy in the sample.The first and second threshold values can be the same or they can bedifferent. In step (c) of this method, the comparison of the firstnormalizing value for said chromosome of interest to a threshold valueindicates the presence or absence of an aneuploidy for said chromosomeof interest, and the comparison of the second normalizing value for saidchromosome of interest to a threshold value verifies the determinationof the presence or absence of an aneuploidy for the chromosome ofinterest. In some embodiments, the first normalizing value is a firstchromosome dose, which is a ratio of the number of sequence tags for thechromosome of interest and a first normalizing chromosome, and thesecond normalizing value is a second chromosome dose, which is a ratioof the number of sequence tags for the chromosome of interest and asecond normalizing chromosome. Optionally, the first and secondnormalizing values can be expressed as normalized chromosome values(NCV) as described herein.

In some other embodiments, the method verifies the determination of thepresence or absence of an aneuploidy of a chromosome of interest in amaternal test sample comprising fetal and maternal nucleic acidmolecules by: (a) obtaining sequence information for the fetal andmaternal nucleic acids in the sample to identify a number of mappedsequence tags for a chromosome of interest and a number of sequence tagsfor at least two normalizing chromosomes, wherein obtaining the sequenceinformation comprises next generation sequencing (NGS); comprisessequencing-by-synthesis using reversible dye terminators; comprisessequencing-by-ligation; or comprises single molecule sequencing; (b)using the number of tags for the chromosome of interest and the numberof tags for a first normalizing chromosome to determine a firstnormalizing value for the chromosome of interest, and using the numberof sequence tags for the first normalizing chromosome and the number ofsequence tags for a second normalizing chromosome to determine a secondnormalizing value for the first normalizing chromosome; and (c)comparing the first normalizing value for the chromosome of interest toa first threshold value and comparing the second normalizing value forthe first normalizing chromosome to a second threshold value todetermine the presence or absence of a fetal aneuploidy in the sample.The first and second threshold values can be the same or they can bedifferent. In step (c) of this method, the comparison of the firstnormalizing value for said chromosome of interest to a threshold valueindicates the presence or absence of an aneuploidy for said chromosomeof interest, and the comparison of the second normalizing value for saidfirst normalizing chromosome to a threshold value verifies thedetermination of the presence or absence of an aneuploidy for thechromosome of interest. In some embodiments, the first normalizing valueis a first chromosome dose, which is a ratio of the number of sequencetags for said chromosome of interest and a first normalizing chromosome,and the second normalizing value a second chromosome dose, which is aratio of the number of sequence tags for the first normalizingchromosome and a second normalizing chromosome. Optionally, the firstand second normalizing values can be expressed as normalized chromosomevalues (NCV) calculated as described herein.

In some embodiments, the first normalizing value is a first chromosomedose, which is a ratio of the number of sequence tags for saidchromosome of interest and a first normalizing chromosome, and thesecond normalizing value a second chromosome dose, which is a ratio ofthe number of sequence tags for the first normalizing chromosome and asecond normalizing chromosome. Optionally, the first and secondnormalizing values can be expressed as normalized chromosome values(NCV) as described herein.

Samples

The sample comprising the mixture of nucleic acids to which the methodsdescribed herein are applied is a biological sample such as a tissuesample, a biological fluid sample, or a cell sample. In someembodiments, the mixture of nucleic acids is purified or isolated fromthe biological sample by any one of the known methods. A sample canconsist of purified or isolated polynucleotide, or it can comprise abiological sample such as a tissue sample, a biological fluid sample, ora cell sample. A biological fluid includes, as non-limiting examples,blood, plasma, serum, sweat, tears, sputum, urine, sputum, ear flow,lymph, saliva, cerebrospinal fluid, ravages, bone marrow suspension,vaginal flow, transcervical lavage, brain fluid, ascites, milk,secretions of the respiratory, intestinal and genitourinary tracts,amniotic fluid, and leukophoresis samples. In some embodiments, thesample is a sample that is easily obtainable by non-invasive procedurese.g. blood, plasma, serum, sweat, tears, sputum, urine, sputum, earflow, and saliva. Preferably, the biological sample is a peripheralblood sample, or the plasma and serum fractions. In other embodiments,the biological sample is a swab or smear, a biopsy specimen, or a cellculture. In another embodiment, the sample is a mixture of two or morebiological samples e.g. a biological sample can comprise two or more ofa biological fluid sample, a tissue sample, and a cell culture sample.As used herein, the terms “blood,” “plasma” and “serum” expresslyencompass fractions or processed portions thereof. Similarly, where asample is taken from a biopsy, swab, smear, etc., the “sample” expresslyencompasses a processed fraction or portion derived from the biopsy,swab, smear, etc.

In some embodiments, samples can be obtained from sources, including,but not limited to, samples from different individuals, differentdevelopmental stages of the same or different individuals, differentdiseased individuals (e.g., individuals with cancer or suspected ofhaving a genetic disorder), normal individuals, samples obtained atdifferent stages of a disease in an individual, samples obtained from anindividual subjected to different treatments for a disease, samples fromindividuals subjected to different environmental factors, or individualswith predisposition to a pathology, or individuals with exposure to aninfectious disease agent (e.g., HIV), and individuals who are recipientsof donor cells, tissues and/or organs. In some embodiments, the sampleis a sample comprising a mixture of different source samples derivedfrom the same or different subjects. For example, a sample can comprisea mixture of cells derived from two or more individuals, as is oftenfound at crime scenes. In one embodiment, the sample is a maternalsample that is obtained from a pregnant female, for example a pregnantwoman. In this instance, the sample can be analyzed using the methodsdescribed herein to provide a prenatal diagnosis of potentialchromosomal abnormalities in the fetus. The maternal sample can be atissue sample, a biological fluid sample, or a cell sample. A biologicalfluid includes, as non-limiting examples, blood, plasma, serum, sweat,tears, sputum, urine, sputum, ear flow, lymph, saliva, cerebrospinalfluid, ravages, bone marrow suspension, vaginal flow, transcervicallavage, brain fluid, ascites, milk, secretions of the respiratory,intestinal and genitourinary tracts, and leukophoresis samples. In someembodiments, the sample is a sample that is easily obtainable bynon-invasive procedures e.g. blood, plasma, serum, sweat, tears, sputum,urine, sputum, ear flow, and saliva. In some embodiments, the biologicalsample is a peripheral blood sample, or the plasma and serum fractions.In other embodiments, the biological sample is a swab or smear, a biopsyspecimen, or a cell culture. In another embodiment, the maternal sampleis a mixture of two or more biological samples e.g. a biological samplecan comprise two or more of a biological fluid sample, a tissue sample,and a cell culture sample. As disclosed above, the terms “blood,”“plasma” and “serum” expressly encompass fractions or processed portionsthereof. Similarly, where a sample is taken from a biopsy, swab, smear,etc., the “sample” expressly encompasses a processed fraction or portionderived from the biopsy, swab, smear, etc.

Samples can also be obtained from in vitro cultured tissues, cells, orother polynucleotide-containing sources. The cultured samples can betaken from sources including, but not limited to, cultures (e.g., tissueor cells) maintained in different media and conditions (e.g., pH,pressure, or temperature), cultures (e.g., tissue or cells) maintainedfor different periods of length, cultures (e.g., tissue or cells)treated with different factors or reagents (e.g., a drug candidate, or amodulator), or cultures of different types of tissue or cells.

Methods of isolating nucleic acids from biological sources are wellknown and will differ depending upon the nature of the source. One ofskill in the art can readily isolate nucleic acid from a source asneeded for the method described herein. In some instances, it can beadvantageous to fragment the nucleic acid molecules in the nucleic acidsample. Fragmentation can be random, or it can be specific, as achieved,for example, using restriction endonuclease digestion. Methods forrandom fragmentation are well known in the art, and include, forexample, limited DNAse digestion, alkali treatment and physicalshearing. In one embodiment, sample nucleic acids are obtained from ascfDNA, which is not subjected to fragmentation. In other embodiments,the sample nucleic acids are obtained as genomic DNA, which is subjectedto fragmentation into fragments of approximately 500 or more base pairs,and to which NGS methods can be readily applied.

Samples that are used for determining a CNV e.g. chromosomal and partialaneuploidies, comprise genomic nucleic acids that are present in cellsi.e. cellular, or that are “cell-free”. Genomic nucleic acids includeDNA and RNA. Preferably, genomic nucleic acids are cellular and/orcfDNA. In some embodiments, the genomic nucleic acid of the sample iscellular DNA, which can be derived from whole cells by manually ormechanically extracting the genomic DNA from whole cells of the same orof differing genetic compositions. Cellular DNA can be derived forexample, from whole cells of the same genetic composition derived fromone subject, from a mixture of whole cells of different subjects, orfrom a mixture of whole cells that differ in genetic composition thatare derived from one subject. Methods for extracting genomic DNA fromwhole cells are known in the art, and differ depending upon the natureof the source. In some embodiments, it can be advantageous to fragmentthe cellular genomic DNA. Fragmentation can be random, or it can bespecific, as achieved, for example, using restriction endonucleasedigestion. Methods for random fragmentation are well known in the art,and include, for example, limited DNAse digestion, alkali treatment, andphysical shearing. In some embodiments, sample nucleic acids areobtained as cellular genomic DNA, which is subjected to fragmentationinto fragments of approximately 500 or more base pairs, which can besequenced by next generation sequencing (NGS).

In some embodiments, cellular genomic DNA is obtained to identifychromosomal aneuploidies of a sample comprising a single genome. Forexample, cellular genomic DNA can be obtained from a sample thatcontains only cells of a pregnant female i.e. the sample is free offetal genomic sequences. Identification of chromosomal aneuploidies froma single genome e.g. maternal only genome, can be used in a comparisonwith chromosomal aneuploidies and/or polymorphisms identified in amixture of fetal and maternal genomes present in maternal plasma toidentify the fetal chromosomal aneuploidies. Similarly, cellular genomicDNA can be obtained from a patient e.g. a cancer patient, at differentstages of treatment to assess the efficacy of the therapeutic regimen byanalyzing possible changes in chromosomal aneuploidies and/orpolymorphisms in the sample DNA

In some embodiments, it is advantageous to obtain cell-free nucleicacids e.g. cell-free DNA (cfDNA). Cell-free nucleic acids, includingcell-free DNA, can be obtained by various methods known in the art frombiological samples including but not limited to plasma, serum and urine(Fan et al., Proc Natl Acad Sci 105:16266-16271 [2008]; Koide et al.,Prenatal Diagnosis 25:604-607 [2005]; Chen et al., Nature Med. 2:1033-1035 [1996]; Lo et al., Lancet 350: 485-487 [1997]; Botezatu etal., Clin Chem. 46: 1078-1084, 2000; and Su et al., J. Mol. Diagn. 6:101-107 [2004]). To separate cfDNA from cells, fractionation,centrifugation (e.g., density gradient centrifugation), DNA-specificprecipitation, or high-throughput cell sorting and/or separation methodscan be used. Commercially available kits for manual and automatedseparation of cfDNA are available (Roche Diagnostics, Indianapolis,Ind., Qiagen, Valencia, Calif., Macherey-Nagel, Duren, Del.). Biologicalsamples comprising cfDNA have been used in assays to determine thepresence or absence of chromosomal abnormalities e.g. trisomy 21, bysequencing assays that can determine chromosomal aneuploidies and/orvarious polymorphisms.

The cfDNA present in the sample can be enriched specifically ornon-specifically prior to preparing a sequencing library. Non-specificenrichment of sample DNA refers to the whole genome amplification of thegenomic DNA fragments of the sample that can be used to increase thelevel of the sample DNA prior to preparing a cfDNA sequencing library.Non-specific enrichment can be the selective enrichment of one of thetwo genomes present in a sample that comprises more than one genome. Forexample, non-specific enrichment can be selective of the fetal genome ina maternal sample, which can be obtained by known methods to increasethe relative proportion of fetal to maternal DNA in a sample.Alternatively, non-specific enrichment can be the non-selectiveamplification of both genomes present in the sample. For example,non-specific amplification can be of fetal and maternal DNA in a samplecomprising a mixture of DNA from the fetal and maternal genomes. Methodsfor whole genome amplification are known in the art. Degenerateoligonucleotide-primed PCR (DOP), primer extension PCR technique (PEP)and multiple displacement amplification (MDA), are examples of wholegenome amplification methods. In some embodiments, the sample comprisingthe mixture of cfDNA from different genomes is unenriched for cfDNA ofthe genomes present in the mixture. In other embodiments, the samplecomprising the mixture of cfDNA from different genomes isnon-specifically enriched for any one of the genomes present in thesample.

Applications

Cell-free fetal DNA and RNA circulating in maternal blood can be usedfor the early non-invasive prenatal diagnosis (NIPD) of an increasingnumber of genetic conditions, both for pregnancy management and to aidreproductive decision-making. The presence of cell-free DNA circulatingin the bloodstream has been known for over 50 years. More recently,presence of small amounts of circulating fetal DNA was discovered in thematernal bloodstream during pregnancy (Lo et al., Lancet 350:485-487[1997]). Thought to originate from dying placental cells, cell-freefetal DNA (cfDNA) has been shown to consists of short fragmentstypically fewer than 200 bp in length Chan et al., Clin Chem 50:88-92[2004]), which can be discerned as early as 4 weeks gestation (Blanes etal., Early Human Dev 83:563-566 [2007]), and known to be cleared fromthe maternal circulation within hours of delivery (Lo et al., Am J HumGenet 64:218-224 [1999]). In addition to cfDNA, fragments of cell-freefetal RNA (cfRNA) can also be discerned in the maternal bloodstream,originating from genes that are transcribed in the fetus or placenta.The extraction and subsequent analysis of these fetal genetic elementsfrom a maternal blood sample offers novel opportunities for NIPD.

The method can be used to determine the presence or absence of a fetalchromosomal aneuploidy in a maternal sample comprising fetal andmaternal nucleic acid molecules e.g. cfDNA. The present method is apolymorphism-independent method for use in NIPD and does not requirethat the fetal cfDNA be distinguished from the maternal cfDNA to enablethe determination of a fetal aneuploidy.

In some embodiments, the sample is a biological fluid sample e.g. ablood sample or fractions thereof. Preferably, the biological sample isselected from plasma, serum and urine. In some embodiments, the maternalsource sample is a peripheral blood sample. In other embodiments, thematernal source sample is a plasma sample. Sequencing of fetal andmaternal nucleic acids can be achieved by any one of the massivelyparallel NGS sequencing methods. In one embodiment, sequencing ismassively parallel sequencing is of clonally amplified cfDNA moleculesor of single cfDNA molecules. In another embodiment, sequencing is saidmassively parallel sequencing is massively parallelsequencing-by-synthesis with reversible dye terminators. In anotherembodiment, sequencing is massively parallel sequencing is performedusing massively parallel sequencing-by-ligation.

In some embodiments, the method can determine or verify the presence orabsence of at least two different chromosomal aneuploidies. In oneembodiment, the method determines the presence or absence of at leasttwo different fetal chromosomal aneuploidies by repeating the steps(a)-(c) for at least two chromosomes of interest, wherein the stepscomprise (a) obtaining sequence information for the fetal and maternalnucleic acids in the maternal sample to identify a number of sequencetags for a chromosome of interest and a number of sequence tags for atleast two normalizing chromosomes; (b) using the number of sequence tagsto calculate a first and a second normalizing value for the chromosomeof interest; and (c) comparing the first normalizing value for thechromosome of interest to a first threshold value and comparing thesecond normalizing value for the chromosome of interest to a secondthreshold value to determine the presence or absence of a fetalaneuploidy in the sample. The first and second threshold values can bethe same or they can be different. In step (c) of this method, thecomparison of the first normalizing value for said chromosome ofinterest to a threshold value indicates the presence or absence of ananeuploidy for said chromosome of interest, and the comparison of thesecond normalizing value for said chromosome of interest to a thresholdvalue verifies the determination of the presence or absence of ananeuploidy for the chromosome of interest. In some embodiments, thefirst normalizing value is a first chromosome dose, which is a ratio ofthe number of sequence tags for the chromosome of interest and a firstnormalizing chromosome, and the second normalizing value is a secondchromosome dose, which is a ratio of the number of sequence tags for thechromosome of interest and a second normalizing chromosome. Optionally,the first and second normalizing values can be expressed as normalizedchromosome values (NCV) as described herein.

Alternatively, the method determines the presence or absence of at leasttwo different fetal chromosomal aneuploidies by repeating the steps(a)-(c) for at least two chromosomes of interest, wherein the stepscomprise (a) obtaining sequence information for the fetal and maternalnucleic acids in the sample to identify a number of mapped sequence tagsfor a chromosome of interest and a number of sequence tags for at leasttwo normalizing chromosomes; (b) using the number of tags for thechromosome of interest and the number of tags for a first normalizingchromosome to determine a first normalizing value for the chromosome ofinterest, and using the number of sequence tags for the firstnormalizing chromosome and the number of sequence tags for a secondnormalizing chromosome to determine a second normalizing value for thefirst normalizing chromosome; and (c) comparing the first normalizingvalue for the chromosome of interest to a first threshold value andcomparing the second normalizing value for the first normalizingchromosome to a second threshold value to determine the presence orabsence of a fetal aneuploidy in the sample. The first and secondthreshold values can be the same or they can be different. In step (c)of this method, the comparison of the first normalizing value for saidchromosome of interest to a threshold value indicates the presence orabsence of an aneuploidy for said chromosome of interest, and thecomparison of the second normalizing value for said first normalizingchromosome to a threshold value verifies the determination of thepresence or absence of an aneuploidy for the chromosome of interest. Insome embodiments, the first normalizing value is a first chromosomedose, which is a ratio of the number of sequence tags for saidchromosome of interest and a first normalizing chromosome, and thesecond normalizing value a second chromosome dose, which is a ratio ofthe number of sequence tags for the first normalizing chromosome and asecond normalizing chromosome. Optionally, the first and secondnormalizing values can be expressed as normalized chromosome values(NCV) as described herein.

In these embodiments, the method can be repeated for all chromosomes todetermine the presence or absence of a fetal chromosomal aneuploidy.

Examples of one or at least two different chromosomal aneuploidies thatcan be determined include T21, T13, T18, T2, T9, and monosomy X. In someembodiments, the maternal sample is obtained from a pregnant woman. Insome embodiments, the maternal sample is a biological fluid sample e.g.a blood sample or the plasma fraction derived therefrom. In someembodiments, the maternal sample is a plasma sample. In someembodiments, the nucleic acids in the maternal sample are cfDNAmolecules.

Examples of fetal chromosomal aneuploidies include without limitationcomplete chromosomal trisomies or monosomies, or partial trisomies ormonosomies. Examples of complete fetal trisomies include trisomy 21(T21; Down Syndrome), trisomy 18 (T18; Edward's Syndrome), trisomy 16(T16), trisomy 22 (T22; Cat Eye Syndrome), trisomy 15 (T15), trisomy 13(T13; Patau Syndrome), trisomy 8 (T8; Warkany Syndrome), trisomy 9 (T9),trisomy 2, and the XXY (Kleinefelter Syndrome), XYY, or XXX trisomies.Examples of partial trisomies include 1q32-44, trisomy 9 p with trisomy,trisomy 4 mosaicism, trisomy 17p, partial trisomy 4q26-qter, trisomy 9,partial 2p trisomy, partial trisomy 1q, and/or partial trisomy6p/monosomy 6q. Examples of fetal monosomies include chromosomalmonosomy X, and partial monosomies of chromosome 13, chromosome 15,chromosome 16, chromosome 18, chromosome 21, and chromosome 22, whichare known to be involved in pregnancy miscarriage. Partial monosomy ofchromosomes typically involved in complete aneuploidy can also bedetermined by the method of the invention. Monosomy 18p is a rarechromosomal disorder in which all or part of the short arm (p) ofchromosome 18 is deleted (monosomic). The disorder is typicallycharacterized by short stature, variable degrees of mental retardation,speech delays, malformations of the skull and facial (craniofacial)region, and/or additional physical abnormalities. Associatedcraniofacial defects may vary greatly in range and severity from case tocase. Conditions caused by changes in the structure or number of copiesof chromosome 15 include Angelman Syndrome and Prader-Willi Syndrome,which involve a loss of gene activity in the same part of chromosome 15,the 15q11-q13 region. It will be appreciated that several translocationsand microdeletions can be asymptomatic in the carrier parent, yet cancause a major genetic disease in the offspring. For example, a healthymother who carries the 15q11-q13 microdeletion can give birth to a childwith Angelman syndrome, a severe neurodegenerative disorder. Thus, thepresent invention can be used to identify such a deletion in the fetus.Partial monosomy 13q is a rare chromosomal disorder that results when apiece of the long arm (q) of chromosome 13 is missing (monosomic).Infants born with partial monosomy 13q may exhibit low birth weight,malformations of the head and face (craniofacial region), skeletalabnormalities (especially of the hands and feet), and other physicalabnormalities. Mental retardation is characteristic of this condition.The mortality rate during infancy is high among individuals born withthis disorder. Almost all cases of partial monosomy 13q occur randomlyfor no apparent reason (sporadic). 22q11.2 deletion syndrome, also knownas DiGeorge syndrome, is a syndrome caused by the deletion of a smallpiece of chromosome 22. The deletion (22 q11.2) occurs near the middleof the chromosome on the long arm of one of the pair of chromosome. Thefeatures of this syndrome vary widely, even among members of the samefamily, and affect many parts of the body. Characteristic signs andsymptoms may include birth defects such as congenital heart disease,defects in the palate, most commonly related to neuromuscular problemswith closure (velo-pharyngeal insufficiency), learning disabilities,mild differences in facial features, and recurrent infections.Microdeletions in chromosomal region 22q11.2 are associated with a 20 to30-fold increased risk of schizophrenia. In one embodiment, the methodof the invention is used to determine partial monosomies including butnot limited to monosomy 18p, partial monosomy of chromosome 15(15q11-q13), partial monosomy 13q, and partial monosomy of chromosome 22can also be determined using the method.

In some embodiments, the chromosomal aneuploidy is a completechromosomal aneuploidy that occurs in a mosaic state. For example, insome embodiments, the chromosomal aneuploidy is an aneuploidy occurringas a true chromosomal mosaicism, wherein the fetal cells can comprisetwo different karyotypes. In other embodiments, the chromosomalaneuploidy is associated with a mosaicism confined predominantly to theplacental tissues. Confined placental mosaicism (CPM) represents adiscrepancy between the chromosomal makeup of the cells in the placentaand the cells in the baby. Most commonly when CPM is found it representsa trisomic cell line in the placenta and a normal diploid chromosomecomplement in the baby. However, the fetus is involved in about 10% ofcases. It is thought that the presence of significant numbers ofabnormal cells in the placenta interferes with proper placentalfunction. An impaired placenta cannot support the pregnancy and this maylead to the loss of a chromosomally normal baby (Tyson & Kalousek,1992). For many of the autosomal trisomies, only mosaic cases survive toterm. For example, complete trisomy 2 contributes significantly to firsttrimester pregnancy losses, occurring in 0.16% of clinically recognizedpregnancies. Trisomy 2 seems to only be compatible with life in a mosaicstate and if the trisomy is confined predominantly to placental tissues.Mosaic trisomy 2 presents one of the more difficult counselingsituations despite that a number of cases of prenatally determinedtrisomy 2 mosaicism have been identified. Outcome ranges from normal toneonatal death. Oligohydramnios (low amniotic fluid) and poorintrauterine growth were the most common features. Abnormal outcome isprobably predominantly a consequence of high levels of trisomy in theplacenta as well as possible presence of low level trisomy in the babyitself. Some uncommon trisomies e.g. trisomy 9, can occur in a mosaic ornon-mosaic state and present with a distinct clinical picture. Thefinding of mosaic trisomy 9 on chorionic villus sampling presents adifficult counseling situation. Diagnosis of trisomy 9 on CVS should befollowed up with amniocentesis and serial ultrasound to exclude trisomyin the fetus, which result in symptoms including dysmorphisms in theskull, nervous system, and mental retardation. Dysmorphisms in theheart, kidneys, and musculoskeletal system may also occur. In most caseswhere trisomy is found on CVS but not on amniocentesis, the outcome isnormal. However, an abnormal outcome can also occur. Despite that anumber of cases of prenatally determined trisomy 9 mosaicism have beenidentified, outcome ranges from normal to neonatal death. Some trisomiesare rare and lethal, while others are viable if confined to theplacental cells. In the latter cases determination of a trisomy, can befollowed up with additional test e.g. amniocentesis, to rule out thatthe trisomy is a fetal trisomy.

The present method is also applicable for determining any chromosomalabnormality if one of the parents is a known carrier of suchabnormality. These include, but not limited to, mosaic for a smallsupernumerary marker chromosome (SMC); t(11;14)(p15;p13) translocation;unbalanced translocation t(8;11)(p23.2;p15.5); 11q23 microdeletion;Smith-Magenis syndrome 17p11.2 deletion; 22q13.3 deletion; Xp22.3microdeletion; 10p14 deletion; 20p microdeletion, DiGeorge syndrome[del(22)(q11.2q11.23)], Williams syndrome (7q11.23 and 7q36 deletions);1p36 deletion; 2p microdeletion; neurofibromatosis type 1 (17q11.2microdeletion), Yq deletion; Wolf-Hirschhorn syndrome (WHS, 4p16.3microdeletion); 1p36.2 microdeletion; 11q14 deletion; 19q13.2microdeletion; Rubinstein-Taybi (16 p13.3 microdeletion); 7p21microdeletion; Miller-Dieker syndrome (17p13.3), 17p11.2 deletion; and2q37 microdeletion.

The method can also be combined with assays for determining otherprenatal conditions associated with the mother and/or the fetus. Themethod is also applicable to determining copy number variations (CNV) ofany sequence of interest in samples comprising mixtures of genomicnucleic acids derived from at least two different genomes and which areknown or are suspected to differ in the amount of one or more sequenceof interest. In some embodiments, the method can be used to determinethe presence or absence of a chromosomal aneuploidy in pregnancies withtwin fetuses (see Example 1). In pregnancies with non-identical twins,the method can determine the presence or absence of a chromosomalaneuploidy in a twin pregnancy, and determine whether one or both twinfetuses carry the aneuploidy by establishing a fetal fraction for eachof the twins and comparing it to the fetal fraction associated with theaneuploidy. A first and a second fetal fraction can be determined forthe first and second twin, respectively, by sequencing polymorphicsequences e.g. SNPs in the maternal plasma cfDNA. Each fetal fractioncan be calculated as the ratio of the portion of the major allelecontributed by the mother and the portion of the minor allelecontributed by the fetus. Methods for determining fetal fraction inmaternal plasma cfDNA are described in pending U.S. patent applicationSer. Nos. 12/958,347 entitled “Methods for Determining Fraction of FetalNucleic Acids in Maternal Samples”, 12/958,356 entitled “Simultaneousdetermination of Aneuploidy and Fetal Fraction” both filed on Dec. 1,2010, and 13/009,718 entitled “Identification of polymorphic sequencesin mixtures of genomic DNA by whole genome sequencing” filed Jan. 19,2011, which are incorporated herein by reference in their entirety). Asthe non-identical twins will differ at least at some of the SNP sites,two separate fetal fractions (first and second) can be determined. Giventhe NCV for chromosome 21 for the sample with the twin pregnancy, afetal fraction associated with the aneuploidy can be estimated as apercent of the difference between the chromosome dose for the aneuploidtwin sample and the average of the chromosome 21 dose in the qualifiedsamples of the training set i.e. NCV chromosome 21 dose in testsample-NCV average chromosome 21 dose in qualified samples/NCVchromosome 21 dose in test sample. The fraction associated with theaneuploidy and calculated suing the NCV for chromosome 21, willcorrespond to one the first or second fetal fractions that weredetermined using differences in SNP sequences, thereby identifyingwhether one or both twins carry the aneuploidy.

In addition to the applicability of the method for determiningchromosomal aneuploidies indicative of a genetic condition in a fetus,the method can be applied determinations of the presence or absence ofchromosomal abnormalities indicative of a disease e.g. cancer, and/orthe status of a disease, determinations of the presence or absence ofnucleic acids of a pathogen e.g. virus, determination of chromosomalabnormalities associated with graft versus host disease (GVHD), anddeterminations of the contribution of individuals in forensic analyses.

CNV in the human genome significantly influence human diversity andpredisposition to disease (Redon et al., Nature 23:444-454 [2006],Shaikh et al. Genome Res 19:1682-1690 [2009]). CNVs have been known tocontribute to genetic disease through different mechanisms, resulting ineither imbalance of gene dosage or gene disruption in most cases. Inaddition to their direct correlation with genetic disorders, CNVs areknown to mediate phenotypic changes that can be deleterious. Recently,several studies have reported an increased burden of rare or de novoCNVs in complex disorders such as Autism, ADHD, and schizophrenia ascompared to normal controls, highlighting the potential pathogenicity ofrare or unique CNVs (Sebat et al., 316:445-449 [2007]; Walsh et al.,Science 320:539-543 [2008]). CNV arise from genomic rearrangements,primarily owing to deletion, duplication, insertion, and unbalancedtranslocation events.

Embodiments of the invention provide for a method to assess copy numbervariation of a sequence of interest e.g. a clinically-relevant sequence,in a test sample that comprises a mixture of nucleic acids derived fromtwo different genomes, and which are known or are suspected to differ inthe amount of one or more sequence of interest. The mixture of nucleicacids is derived from two or more types of cells. In one embodiment, themixture of nucleic acids is derived from normal and cancerous cellsderived from a subject suffering from a medical condition e.g. cancer.

It is believed that many solid tumors, such as breast cancer, progressfrom initiation to metastasis through the accumulation of severalgenetic aberrations. [Sato et al., Cancer Res., 50: 7184-7189 [1990];Jongsma et al., J Clin PAthol: Mol Path 55:305-309 [2002])]. Suchgenetic aberrations, as they accumulate, may confer proliferativeadvantages, genetic instability and the attendant ability to evolve drugresistance rapidly, and enhanced angiogenesis, proteolysis andmetastasis. The genetic aberrations may affect either recessive “tumorsuppressor genes” or dominantly acting oncogenes. Deletions andrecombination leading to loss of heterozygosity (LOH) are believed toplay a major role in tumor progression by uncovering mutated tumorsuppressor alleles.

cfDNA has been found in the circulation of patients diagnosed withmalignancies including but not limited to lung cancer (Pathak et al.Clin Chem 52:1833-1842 [2006]), prostate cancer (Schwartzenbach et al.Clin Cancer Res 15:1032-8 [2009]), and breast cancer (Schwartzenbach etal. available online at breast-cancer-research.com/content/11/5/R71[2009]). Identification of genomic instabilities associated with cancersthat can be determined in the circulating cfDNA in cancer patients is apotential diagnostic and prognostic tool. In one embodiment, the methodof the invention assesses CNV of a sequence of interest in a samplecomprising a mixture of nucleic acids derived from a subject that issuspected or is known to have cancer e.g. carcinoma, sarcoma, lymphoma,leukemia, germ cell tumors and blastoma. In one embodiment, the sampleis a plasma sample derived (processes) from peripheral blood and thatcomprises a mixture of cfDNA derived from normal and cancerous cells. Inanother embodiment, the biological sample that is needed to determinewhether a CNV is present is derived from a mixture of cancerous andnon-cancerous cells from other biological fluids including but notlimited to serum, sweat, tears, sputum, urine, sputum, ear flow, lymph,saliva, cerebrospinal fluid, ravages, bone marrow suspension, vaginalflow, transcervical lavage, brain fluid, ascites, milk, secretions ofthe respiratory, intestinal and genitourinary tracts, and leukophoresissamples, or in tissue biopsies, swabs or smears.

The sequence of interest is a nucleic acid sequence that is known or issuspected to play a role in the development and/or progression of thecancer. Examples of a sequence of interest include nucleic acidssequences that are amplified or deleted in cancerous cells as describedin the following.

Dominantly acting genes associated with human solid tumors typicallyexert their effect by overexpression or altered expression. Geneamplification is a common mechanism leading to upregulation of geneexpression. Evidence from cytogenetic studies indicates that significantamplification occurs in over 50% of human breast cancers. Most notably,the amplification of the proto-oncogene human epidermal growth factorreceptor 2 (HER2) located on chromosome 17 (17(17q21-q22)), results inoverexpression of HER2 receptors on the cell surface leading toexcessive and dysregulated signaling in breast cancer and othermalignancies (Park et al., Clinical Breast Cancer 8:392-401 [2008]). Avariety of oncogenes have been found to be amplified in other humanmalignancies. Examples of the amplification of cellular oncogenes inhuman tumors include amplifications of: c-myc in promyelocytic leukemiacell line HL60, and in small-cell lung carcinoma cell lines, N-myc inprimary neuroblastomas (stages III and IV), neuroblastoma cell lines,retinoblastoma cell line and primary tumors, and small-cell lungcarcinoma lines and tumors, L-myc in small-cell lung carcinoma celllines and tumors, c-myb in acute myeloid leukemia and in colon carcinomacell lines, c-erbb in epidermoid carcinoma cell, and primary gliomas,c-K-ras-2 in primary carcinomas of lung, colon, bladder, and rectum,N-ras in mammary carcinoma cell line (Varmus H., Ann Rev Genetics 18:553-612 (1984) [cited in Watson et al., Molecular Biology of the Gene(4th ed.; Benjamin/Cummings Publishing Co. 1987)].

Chromosomal deletions involving tumor suppressor genes may play animportant role in the development and progression of solid tumors. Theretinoblastoma tumor suppressor gene (Rb-1), located in chromosome13q14, is the most extensively characterized tumor suppressor gene. TheRb-1 gene product, a 105 kDa nuclear phosphoprotein, apparently plays animportant role in cell cycle regulation (Howe et al., Proc Natl Acad Sci(USA) 87:5883-5887 [1990]). Altered or lost expression of the Rb proteinis caused by inactivation of both gene alleles either through a pointmutation or a chromosomal deletion. Rb-i gene alterations have beenfound to be present not only in retinoblastomas but also in othermalignancies such as osteosarcomas, small cell lung cancer (Rygaard etal., Cancer Res 50: 5312-5317 [1990)]) and breast cancer. Restrictionfragment length polymorphism (RFLP) studies have indicated that suchtumor types have frequently lost heterozygosity at 13q suggesting thatone of the Rb-1 gene alleles has been lost due to a gross chromosomaldeletion (Bowcock et al., Am J Hum Genet, 46: 12 [1990]). Chromosome 1abnormalities including duplications, deletions and unbalancedtranslocations involving chromosome 6 and other partner chromosomesindicate that regions of chromosome 1, in particular 1q21-1q32 and1p11-13, might harbor oncogenes or tumor suppressor genes that arepathogenetically relevant to both chronic and advanced phases ofmyeloproliferative neoplasms (Caramazza et al., Eur J Hematol 84:191-200[2010]). Myeloproliferative neoplasms are also associated with deletionsof chromosome 5. Complete loss or interstitial deletions of chromosome 5are the most common karyotypic abnormality in myelodysplastic syndromes(MDSs). Isolated del(5q)/5q-MDS patients have a more favorable prognosisthan those with additional karyotypic defects, who tend to developmyeloproliferative neoplasms (MPNs) and acute myeloid leukemia. Thefrequency of unbalanced chromosome 5 deletions has led to the idea that5q harbors one or more tumor-suppressor genes that have fundamentalroles in the growth control of hematopoietic stem/progenitor cells(HSCs/HPCs). Cytogenetic mapping of commonly deleted regions (CDRs)centered on 5q31 and 5q32 identified candidate tumor-suppressor genes,including the ribosomal subunit RPS14, the transcription factorEgr1/Krox20 and the cytoskeletal remodeling protein, alpha-catenin(Eisenmann et al., Oncogene 28:3429-3441 [2009]). Cytogenetic andallelotyping studies of fresh tumours and tumour cell lines have shownthat allelic loss from several distinct regions on chromosome 3p,including 3p25, 3p21-22, 3p21.3, 3p12-13 and 3p14, are the earliest andmost frequent genomic abnormalities involved in a wide spectrum of majorepithelial cancers of lung, breast, kidney, head and neck, ovary,cervix, colon, pancreas, esophagous, bladder and other organs. Severaltumor suppressor genes have been mapped to the chromosome 3p region, andare thought that interstitial deletions or promoter hypermethylationprecede the loss of the 3p or the entire chromosome 3 in the developmentof carcinomas (Angeloni D., Briefings Functional Genomics 6:19-39[2007]).

Newborns and children with Down syndrome (DS) often present withcongenital transient leukemia and have an increased risk of acutemyeloid leukemia and acute lymphoblastic leukemia. Chromosome 21,harboring about 300 genes, may be involved in numerous structuralaberrations, e.g., translocations, deletions, and amplifications, inleukemias, lymphomas, and solid tumors. Moreover, genes located onchromosome 21 have been identified that play an important role intumorigenesis. Somatic numerical as well as structural chromosome 21aberrations are associated with leukemias, and specific genes includingRUNX1, TMPRSS2, and TFF, which are located in 21q, play a role intumorigenesis (Fonatsch C Gene Chromosomes Cancer 49:497-508 [2010]).

In one embodiment, the method provides a means to assess the associationbetween gene amplification and the extent of tumor evolution.Correlation between amplification and/or deletion and stage or grade ofa cancer may be prognostically important because such information maycontribute to the definition of a genetically based tumor grade thatwould better predict the future course of disease with more advancedtumors having the worst prognosis. In addition, information about earlyamplification and/or deletion events may be useful in associating thoseevents as predictors of subsequent disease progression. Geneamplification and deletions as identified by the method can beassociated with other known parameters such as tumor grade, histology,Brd/Urd labeling index, hormonal status, nodal involvement, tumor size,survival duration and other tumor properties available fromepidemiological and biostatistical studies. For example, tumor DNA to betested by the method could include atypical hyperplasia, ductalcarcinoma in situ, stage I-III cancer and metastatic lymph nodes inorder to permit the identification of associations betweenamplifications and deletions and stage. The associations made may makepossible effective therapeutic intervention. For example, consistentlyamplified regions may contain an overexpressed gene, the product ofwhich may be able to be attacked therapeutically (for example, thegrowth factor receptor tyrosine kinase, p185^(HER2)).

The method can be used to identify amplification and/or deletion eventsthat are associated with drug resistance by determining the copy numbervariation of nucleic acids from primary cancers to those of cells thathave metastasized to other sites.” If gene amplification and/or deletionis a manifestation of karyotypic instability that allows rapiddevelopment of drug resistance, more amplification and/or deletion inprimary tumors from chemoresistant patients than in tumors inchemosensitive patients would be expected. For example, if amplificationof specific genes is responsible for the development of drug resistance,regions surrounding those genes would be expected to be amplifiedconsistently in tumor cells from pleural effusions of chemoresistantpatients but not in the primary tumors. Discovery of associationsbetween gene amplification and/or deletion and the development of drugresistance may allow the identification of patients that will or willnot benefit from adjuvant therapy.

Apparatus and Systems for Determining CNV

Analysis of the sequencing data and the determination derived therefromare typically performed using various computer hardware, computeralgorithms and computer programs. The methods of the invention aretherefore typically computer-implemented or computer-assisted methods.

In one embodiment, the invention provides a computer program product forgenerating an output indicating the presence or absence of a fetalaneuploidy in a test sample. The computer product comprises a computerreadable medium having a computer executable logic recorded thereon forenabling a processor to determine the presence or absence of a fetalaneuploidy comprising: a receiving procedure for receiving sequencingdata from at least a portion of nucleic acid molecules from a maternalbiological sample, wherein said sequencing data comprises sequencereads; computer assisted logic for analyzing a fetal aneuploidy fromsaid received data; and an output procedure for generating an outputindicating the presence, absence or kind of said fetal aneuploidy. Themethod of the invention can be performed using a computer-readablemedium having stored thereon computer-readable instructions for carryingout a method for identifying any CNV e.g. chromosomal or partialaneuploidies. In one embodiment, the invention provides acomputer-readable medium having stored thereon computer-readableinstructions for identifying at least one chromosome suspected to beinvolved with a chromosomal aneuploidy e.g. trisomy 21, trisomy, 13,trisomy 18, or monosomy X.

In one embodiment, the invention provides a computer-readable mediumhaving stored thereon computer-readable instructions for carrying out amethod comprising the steps: (a) using sequence information obtainedfrom fetal and maternal nucleic acids in a sample to identify a numberof sequence tags for a chromosome of interest and a number of sequencetags for at least two normalizing chromosomes; (b) using the numbers ofsequence tags to calculate a first normalizing value and a secondnormalizing value for the chromosome of interest; and (c) comparing thefirst normalizing value for the chromosome of interest to a firstthreshold value and comparing the second normalizing value for thechromosome of interest to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. Thecomputer-readable medium may have stored thereon computer-readableinstructions for carrying out a method wherein the first normalizingvalue for the chromosome of interest is a first chromosome dose, thefirst chromosome dose being a ratio of the number of sequence tags forthe chromosome of interest and a first normalizing chromosome, andwherein the second normalizing value for the chromosome of interest is asecond chromosome dose, the second chromosome dose being a ratio of thenumber of sequence tags for the chromosome of interest and a secondnormalizing chromosome.

In one embodiment, the invention provides a computer-readable mediumhaving stored thereon computer-readable instructions for carrying out amethod comprising the steps: (a) using sequence information obtainedfrom fetal and maternal nucleic acids in a sample to identify a numberof sequence tags for a chromosome of interest and a number of sequencetags for at least two normalizing chromosomes; (b) using the number ofsequence tags for the chromosome of interest and the number of sequencetags for a first normalizing chromosome to determine a first normalizingvalue for the chromosome of interest, and using the number of sequencetags for the first normalizing chromosome and the number of sequencetags for a second normalizing chromosome to determine a secondnormalizing value for the first normalizing chromosome; (c) comparingthe first normalizing value for the chromosome of interest to a firstthreshold value and comparing the second normalizing value for the firstnormalizing chromosome to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. Thecomputer-readable medium may have stored thereon computer-readableinstructions for carrying out a method wherein the first normalizingvalue for the chromosome of interest is a first chromosome dose, thefirst chromosome dose being a ratio of the number of sequence tags forthe chromosome of interest and a first normalizing chromosome, andwherein the second normalizing value for the chromosome of interest is asecond chromosome dose, the second chromosome dose being a ratio of thenumber of sequence tags for the first normalizing chromosome and asecond normalizing chromosome.

In one embodiment, the invention provides a computer processing systemwhich is adapted or configured to perform a method according to theinvention. For example, the invention provides a computer processingsystem which is adapted and configured to carry out a method comprisingthe steps: (a) using sequence information obtained from fetal andmaternal nucleic acids in a sample to identify a number of sequence tagsfor a chromosome of interest and a number of sequence tags for at leasttwo normalizing chromosomes; (b) using the numbers of sequence tags tocalculate a first normalizing value and a second normalizing value forthe chromosome of interest; and (c) comparing the first normalizingvalue for the chromosome of interest to a first threshold value andcomparing the second normalizing value for the chromosome of interest toa second threshold value to determine the presence or absence of a fetalaneuploidy in the sample. The computer processing system may be adaptedand configured to carry out a method wherein the first normalizing valuefor the chromosome of interest is a first chromosome dose, the firstchromosome dose being a ratio of the number of sequence tags for thechromosome of interest and a first normalizing chromosome, and whereinthe second normalizing value for the chromosome of interest is a secondchromosome dose, the second chromosome dose being a ratio of the numberof sequence tags for the chromosome of interest and a second normalizingchromosome.

In one embodiment, the invention provides a computer processing systemwhich is adapted and configured to carry out a method comprising thesteps: (a) using sequence information obtained from fetal and maternalnucleic acids in a sample to identify a number of sequence tags for achromosome of interest and a number of sequence tags for at least twonormalizing chromosomes; (b) using the number of sequence tags for thechromosome of interest and the number of sequence tags for a firstnormalizing chromosome to determine a first normalizing value for thechromosome of interest, and using the number of sequence tags for thefirst normalizing chromosome and the number of sequence tags for asecond normalizing chromosome to determine a second normalizing valuefor the first normalizing chromosome; (c) comparing the firstnormalizing value for the chromosome of interest to a first thresholdvalue and comparing the second normalizing value for the firstnormalizing chromosome to a second threshold value to determine thepresence or absence of a fetal aneuploidy in the sample. The computerprocessing system may be adapted and configured to carry out a methodwherein the first normalizing value for the chromosome of interest is afirst chromosome dose, the first chromosome dose being a ratio of thenumber of sequence tags for the chromosome of interest and a firstnormalizing chromosome, and wherein the second normalizing value for thechromosome of interest is a second chromosome dose, the secondchromosome dose being a ratio of the number of sequence tags for thefirst normalizing chromosome and a second normalizing chromosome.

The invention also provides apparatus adapted or configured to perform amethod according to the invention, wherein the apparatus optionallycomprises a sequencing device adapted or configured to sequence fetaland maternal nucleic acid molecules in a sample. For example, theinvention provides apparatus which comprises: (a) a sequencing deviceadapted or configured to sequence fetal and maternal nucleic acidmolecules in a sample using a sequencing method as described herein,thereby generating sequence information; and (b) a computer processingsystem adapted or configured to use the sequence information generatedby the sequencing device in a method as described herein, wherein thecomputer processing system is optionally directly linked to thesequencing device such that the sequence information can beautomatically transferred from the sequencing device to the computerprocessing system. The apparatus may further comprise a transfer deviceadapted or configured to transfer samples to the sequencing device forsequencing.

The present invention is described in further detail in the followingExamples which are not in any way intended to limit the scope of theinvention as claimed. The attached Figures are meant to be considered asintegral parts of the specification and description of the invention.The following examples are offered to illustrate, but not to limit theclaimed invention.

EXAMPLES Example 1 Optimal Determination of Fetal ChromosomalAbnormalities Using Massively Parallel DNA Sequencing of Cell Free FetalDNA from Maternal Blood: Test Set 1 Independent of Training Set 1

The study was conducted by qualified site clinical research personnel at13 US clinic locations between April 2009 and July 2010 under a humansubject protocol approved by institutional review boards (IRBs) at eachinstitution. Informed written consent was obtained from each subjectprior to study participation. The protocol was designed to provide bloodsamples and clinical data to support development of noninvasive prenatalgenetic diagnostic methods. Pregnant women, age 18 years or older wereeligible for inclusion. For patients undergoing clinically indicated CVSor amniocentesis blood was collected prior to performance of theprocedure, and results of fetal karyotype was also collected. Peripheralblood samples (two tubes or ˜20 mL total) were drawn from all subjectsin acid citrate dextrose (ACD) tubes (Becton Dickinson). All sampleswere de-identified and assigned an anonymous patient ID number. Bloodsamples were shipped overnight to the laboratory in temperaturecontrolled shipping containers provided for the study. Time elapsedbetween blood draw and sample receipt was recorded as part of the sampleaccessioning.

Site research coordinators entered clinical data relevant to thepatient's current pregnancy and history into study case report forms(CRFs) using the anonymous patient ID number. Cytogenetic analysis offetal karyotype from invasive prenatal procedure samples was performedper local laboratories and the results were also recorded in study CRFs.All data obtained on CRFs were entered into a clinical database thelaboratory. Cell free plasma was obtained from individual blood tubesutilizing at two-step centrifugation process within 24-48 hours ofsample of venipuncture. Plasma from a single blood tube was sufficientfor sequencing analysis. Cell-free DNA was extracted from cell-freeplasma by using QIAamp DNA Blood Mini kit (Qiagen) according to themanufacturer's instructions. Since the cell free DNA fragments are knownto be approximately 170 base pairs (bp) in length (Fan et al., Clin Chem56:1279-1286 [2010]) no fragmentation of the DNA was required prior tosequencing.

For the training set samples, cfDNA was sent to Prognosys Biosciences,Inc. (La Jolla, Calif.) for sequencing library preparation (cfDNA bluntended and ligated to universal adapters) and sequencing using standardmanufacturer protocols with the Illumina Genome Analyzer IIxinstrumentation. Single-end reads of 36 base pairs were obtained. Uponcompletion of the sequencing, all base call files were collected andanalyzed. For the test set samples, sequencing libraries were preparedand sequencing carried out on Illumina Genome Analyzer IIx instrument.Sequencing library preparation was performed as follows. The full-lengthprotocol described is essentially the standard protocol provided byIllumina, and only differs from the Illumina protocol in thepurification of the amplified library: the Illumina protocol instructsthat the amplified library be purified using gel electrophoresis, whilethe protocol described herein uses magnetic beads for the samepurification step. Approximately 2 ng of purified cfDNA that had beenextracted from maternal plasma was used to prepare a primary sequencinglibrary using NEBNext™ DNA Sample Prep DNA Reagent Set 1 (Part No.E6000L; New England Biolabs, Ipswich, Mass.) for Illumina® essentiallyaccording to the manufacturer's instructions. All steps except for thefinal purification of the adaptor-ligated products, which was performedusing Agencourt magnetic beads and reagents instead of the purificationcolumn, were performed according to the protocol accompanying theNEBNext™ Reagents for Sample Preparation for a genomic DNA library thatis sequenced using the Illumina® GAII. The NEBNext™ protocol essentiallyfollows that provided by Illumina, which is available atgrcf.jhml.edu/hts/protocols/11257047_ChIP_Sample_Prep.pdf.

The overhangs of approximately 2 ng purified cfDNA fragments containedin 40 μl were converted into phosphorylated blunt ends according to theNEBNext® End Repair Module by incubating the 40 μl cfDNA with 5 μl 10×phosphorylation buffer, 2 μl deoxynucleotide solution mix (10 mM eachdNTP), 1 μl of a 1:5 dilution of DNA Polymerase I, 1 μl T4 DNAPolymerase and 1 μl T4 Polynucleotide Kinase provided in the NEBNext™DNA Sample Prep DNA Reagent Set 1 in a 200 μl microfuge tube in athermal cycler for 30 minutes at 20° C. The sample was cooled to 4° C.,and purified using a QIAQuick column provided in the QIAQuick PCRPurification Kit (QIAGEN Inc., Valencia, Calif.) as follows. The 50 μlreaction was transferred to 1.5 ml microfuge tube, and 250 μl of QiagenBuffer PB were added. The resulting 300 μl were transferred to aQIAquick column, which was centrifuged at 13,000 RPM for 1 minute in amicrofuge. The column was washed with 750 μl Qiagen Buffer PE, andre-centrifuged. Residual ethanol was removed by an additionalcentrifugation for 5 minutes at 13,000 RPM. The DNA was eluted in 39 μlQiagen Buffer EB by centrifugation. dA tailing of 34 μl of theblunt-ended DNA was accomplished using 16 μl of the dA-tailing mastermix containing the Klenow fragment (3′ to 5′ exo minus) (NEBNext™ DNASample Prep DNA Reagent Set 1), and incubating for 30 minutes at 37° C.according to the manufacturer's NEBNext® dA-Tailing Module. The samplewas cooled to 4° C., and purified using a column provided in theMinElute PCR Purification Kit (QIAGEN Inc., Valencia, Calif.) asfollows. The 50 μl reaction was transferred to 1.5 ml microfuge tube,and 250 μl of Qiagen Buffer PB were added. The 300 μl were transferredto the MinElute column, which was centrifuged at 13,000 RPM for 1 minutein a microfuge. The column was washed with 750 μl Qiagen Buffer PE, andre-centrifuged. Residual ethanol was removed by an additionalcentrifugation for 5 minutes at 13,000 RPM. The DNA was eluted in 15 μlQiagen Buffer EB by centrifugation. Ten microliters of the DNA eluatewere incubated with 1 μl of a 1:5 dilution of the Illumina GenomicAdapter Oligo Mix (Part No. 1000521), 15 μl of 2× Quick LigationReaction Buffer, and 4 μl Quick T4 DNA Ligase, for 15 minutes at 25° C.according to the NEBNext® Quick Ligation Module. The sample was cooledto 4° C., and purified using a MinElute column as follows. One hundredand fifty microliters of Qiagen Buffer PE were added to the 30 μlreaction, and the entire volume was transferred to a MinElute columnwere transferred to a MinElute column, which was centrifuged at 13,000RPM for 1 minute in a microfuge. The column was washed with 750 μlQiagen Buffer PE, and re-centrifuged. Residual ethanol was removed by anadditional centrifugation for 5 minutes at 13,000 RPM. The DNA waseluted in 28 μl Qiagen Buffer EB by centrifugation. Twenty threemicroliters of the adaptor-ligated DNA eluate were subjected to 18cycles of PCR (98° C. for 30 seconds; 18 cycles of 98° C. for 10seconds, 65° C. for 30 seconds, and 72° C. for 30; final extension at72° C. for 5 minutes, and hold at 4° C.) using Illumina Genomic PCRPrimers (Part Nos. 100537 and 1000538) and the Phusion HF PCR Master Mixprovided in the NEBNext™ DNA Sample Prep DNA Reagent Set 1, according tothe manufacturer's instructions. The amplified product was purifiedusing the Agencourt AMPure XP PCR purification system (AgencourtBioscience Corporation, Beverly, Mass.) according to the manufacturer'sinstructions available on the world wide web atbeckmangenomics.com/products/AMPureXPProtocol_(—000387)v001.pdf. TheAgencourt AMPure XP PCR purification system removes unincorporateddNTPs, primers, primer dimers, salts and other contaminates, andrecovers amplicons greater than 100 bp. The purified amplified productwas eluted from the Agencourt beads in 40 μl of Qiagen EB Buffer and thesize distribution of the libraries was analyzed using the Agilent DNA1000 Kit for the 2100 Bioanalyzer (Agilent technologies Inc., SantaClara, Calif.).

For both the training and test sample sets, single-end reads of 36 basepairs were sequenced.

Data Analysis and Sample Classification

Sequence reads 36 bases in length were aligned to the human genomeassembly hg18 obtained from the UCSC database (available on the worldwide web at hgdownload.cse.ucsc.edu/goldenPath/hg18/bigZips/).Alignments were carried out utilizing the Bowtie short read aligner(version 0.12.5) allowing for up to two base mismatches during alignment(Langmead et al., Genome Biol 10:R25 [2009]. Only reads thatunambiguously mapped to a single genomic location were included. Genomicsites where reads mapped were counted and included in the calculation ofchromosome ratios (see below). Regions on the Y chromosome wheresequence tags from male and female fetuses map without anydiscrimination were excluded from the analysis (specifically, from base0 to base 2×10⁶; base 10×10⁶ to base 13×10⁶; and base 23×10⁶ to the endof chromosome Y).

Intra-run and inter-run sequencing variation in the chromosomaldistribution of sequence reads can obscure the effects of fetalaneuploidy on the distribution of mapped sequence sites. To correct forsuch variation, a chromosome dose was calculated as the count of mappedsites for a given chromosome of interest is normalized to countsobserved on a predetermined normalizing chromosome or a set ofnormalizing chromosomes. The normalizing chromosome or set ofnormalizing chromosomes was first identified in a subset of samples inthe training set of samples that were unaffected i.e. qualified sampleshaving diploid karyotypes for chromosomes of interest 21, 18, 13 and X,considering each autosome as a potential denominator in a ratio ofcounts with our chromosomes of interest. Denominator chromosomes i.e.normalizing chromosomes were selected that minimized the variation ofthe chromosome ratios within and between sequencing runs. Eachchromosome of interest was determined to have a distinct denominator(Table 1).

The chromosome doses for each of the chromosomes of interest in thequalified samples provides a measure of the variation in the totalnumber of mapped sequence tags for each chromosome of interest relativeto that of each of the remaining chromosomes. Thus, qualified chromosomedoses can identify the chromosome or a group of chromosomes i.e.normalizing chromosome that has a variation among samples that isclosest to the variation of the chromosome of interest, and that wouldserve as ideal sequences for normalizing values for further statisticalevaluation.

Chromosome doses for all samples in the training set i.e. qualified andaffected, also serve as the basis for determining threshold values whenidentifying aneuploidies in test samples as described in the following.

TABLE 1 Normalizing Chromosomes for Determining Chromosome DosesChromosome of Interest - Normalizing Chromosome - Chromosome ofNumerator (Chr mapped Denominator (Chr mapped Interest counts) counts)21 Chr 21 Chr 9 18 Chr 18 Chr 8 13 Chr 13 Sum(Chr 2-6) X Chr X Chr 6 YChr Y Sum(Chr 2-6)For each chromosome of interest in each sample in the test set, anormalizing value was determined and used to determine the presence orabsence of an aneuploidy. The normalizing value was calculated as achromosome dose that can be further computed to provide a normalizedchromosome value (NCV).Chromosome Doses

For the test set, a chromosome dose was calculated for each chromosomeof interest, 21, 18, 13, X and Y for every sample. As provided in Tableabove 1, the chromosome dose for chromosome 21 was calculated as a ratioof the number of tags in the test sample that mapped to chromosome 21 inthe test sample, and the number of tags in the test sample that mappedto chromosome 9; the chromosome dose for chromosome 18 was calculated asa ratio of the number of tags in the test sample that mapped tochromosome 18 in the test sample, and the number of tags in the testsample that mapped to chromosome 8; the chromosome dose for chromosome13 was calculated as a ratio of the number of tags in the test samplethat mapped to chromosome 13 in the test sample, and the number of tagsin the test sample that mapped to chromosomes 2-6; the chromosome dosefor chromosome X was calculated as a ratio of the number of tags in thetest sample that mapped to chromosome X in the test sample, and thenumber of tags in the test sample that mapped to chromosome 6; and thechromosome dose for chromosome Y was calculated as a ratio of the numberof tags in the test sample that mapped to chromosome Y in the testsample, and the number of tags in the test sample that mapped tochromosomes 2-6.

Normalized Chromosome Values

Using the chromosome dose for each of the chromosomes of interest ineach of the test samples, and the mean of the corresponding chromosomedose determined in the qualified samples of the training set, anormalized chromosome value (NCV) was calculated using the equation:

${NCV}_{ij} = \frac{x_{ij} - {\hat{\mu}}_{j}}{{\hat{\sigma}}_{j}}$where {circumflex over (μ)}_(j) AND {circumflex over (σ)}_(j) are theestimated training set mean and standard deviation respectively for thej-th chromosome ratio, and x_(ij) is the observed j-th chromosome ratiofor sample i. When chromosome ratios are normally distributed, the NCVis equivalent to a statistical z-score for the ratios. No significantdeparture from linearity is observed in a quantile-quantile plot of theNCVs from unaffected samples. In addition, standard tests of normalityfor the NCVs fail to reject the null hypothesis of normality. For boththe Kolmogrov-Smirnov and Shapiro-Wilk tests the significance value isgreater than 0.05.

For the test set, an NCV was calculated for each chromosome of interest,21, 18, 13, X and Y for every sample. To insure a safe and effectiveclassification scheme, conservative boundaries were chosen foraneuploidy classification. For classification of the autosomes'aneuploidy state, a NCV>4.0 was required to classify the chromosome asaffected (i.e. aneuploid for that chromosome) and a NCV<2.5 to classifya chromosome as unaffected. Samples with autosomes that have an NCVbetween 2.5 and 4.0 were classified as “no call”.

Sex chromosome classification in the test was performed by sequentialapplication of NCVs for both X and Y as follows:

-   -   1. If NCV Y>−2.0 standard deviations from the mean of male        samples, then the sample was classified as male (XY).    -   2. If NCV Y<−2.0 standard deviations from the mean of male        samples, and NCV X>−2.0 standard deviations from the mean of        female samples, then the sample was classified as female (XX).    -   3. If NCV Y<−2.0 standard deviations from the mean of male        samples, and NCV X<−3.0 standard deviations from the mean of        female samples, then the sample was classified as monosomy X,        i.e. Turner syndrome.    -   4. If the NCVs did not fit into any of the above criteria, then        the sample was classified as a “no call” for sex.        Results        Study Population Demographics

A total of 1,014 patients were enrolled between April 2009 and July2010. The patient demographics, invasive procedure type and karyotyperesults are summarized in Table 2. The average age of study participantswas 35.6 yrs (range 17 to 47 yrs) and gestational age ranged between 6weeks, 1 day to 38 weeks, 1 day (mean 15 weeks, 4 days). The overallincidence of abnormal fetal chromosome karyotypes was 6.8% with T21incidence of 2.5%. Of 946 subjects with singleton pregnancies andkaryotype, 906 (96%) showed at least one clinically recognized riskfactor for fetal aneuploidy prior to prenatal procedure. Eveneliminating those with advanced maternal age as their sole indication,the data demonstrates a very high false positive rate for currentscreening modalities. Ultrasound findings of increased nuchaltranslucency, cystic hygroma, or other structural congenital abnormalityby ultrasound were most predictive of abnormal karyotype in this cohort.

TABLE 2 Patient Demographics Total Enrolled Training Set Test Set (N =1014) (N = 71) (N = 48) Dates of Enrollment April 2009-July 2010 April2009-December 2009 January 2010-June 2010 Number enrolled 1014 435 575Maternal Age, yrs Mean (SD)  35.6 (5.66) 36.4 (6.05) 34.2 (8.22) Min/Max17/47 20/46 18/46 Not Specified, N  11  3  0 Ethnicity, N (%) Caucasian 636 (62.7)   50 (70.4)   24 (50.0) Hispanic  167 (16.5)   6 (8.5)   13(27.0) Asian  63 (6.2)   6 (8.5)   5 (10.4) Multi, more than one  53(5.2)   6 (8.5)   1 (2.1) African American  41 (4.0)   1 (1.3)   3 (6.3)Other  36 (3.6)   2 (2.8)   1 (2.1) Native American   9 (0.9)   0 (0.0)  1 (2.1) Not Specified   9 (0.9)   0 (0.0)   0 (0.0) Gestational Age,wks, days Mean 15 w 4 d 14 w 5 d 15 w 3 d Min/Max 6 w 1 d/38 w 1 d 10 w0 d/23 w 1 d 10 w 4 d/28 w 3 d Number of Fetus, N 1  982  67  47 2  30 4  1 3   2  0  0 Prenatal Procedure, N (%) CVS  430 (42.4)   38 (53.5)  28 (58.3) Amniocentesis  571 (56.3)   32 (45.1)   20 (41.7) Notspecified   3 (0.3)   1 (1.4)   0 (0.0) Not performed  10 (1.0)   0(0.0)   0 (0.0) Fetal Karyotype, N (%) 46 XX 453* (43.9)  22* (29.7)  7* (14.6) 46 XY 474* (45.9)  26* (35.1)   14 (29.2) 47, +21, bothsexes  25* (2.4)  10* (13.5)   13 (27.1) 47, +18, both sexes  14 (1.4)  5 (6.8)   8 (16.7) 47, +13, both sexes   4 (0.4)   2 (2.7)   1 (2.1)45, X   8 (0.8)   3 (4.1)   3 (6.3) Complex, other  18* (1.7)   6 (8.1)  2 (4.2) Karyotype not available  36 (3.5)   0 (0.0)   0 (0.0) PrenatalScreening Risks Analyzed for Karyotyped Non-sequenced Training AnalyzedTest Singletons, N (%) N = 834 N = 65 N = 47 AMA only (≧35 years)  445(53.4)   27 (41.5)   21 (44.7) Screen positive (trisomy)**  149 (17.9)  18 (27.7)   9 (19.1) Increased NT  35 (4.2)   3 (4.6)   5 (10.6)Cystic Hygroma  12 (1.4)   5 (7.7)   4 (8.5) Cardiac Defect  14 (1.7)  0 (0.0)   4 (8.5) Other Congenital  78 (9.4)   4 (6.2)   3 (6.4)Abnormality  64 (7.7)   5 (7.7)   1 (2.1) Other Maternal Risk  37 (4.4)  3 (4.6)   0 (0.0) None specified *Includes results of fetuses frommultiple gestations, **Assessed and reported by cliniciansAbbreviations: AMA = Advanced Maternal Age, NT = nuchal translucency

The distribution of diverse ethnic backgrounds represented in this studypopulation is also shown in Table 2. Overall, 63% of the patients inthis study were Caucasian, 17% Hispanic, 6% Asian, 5% multi-ethnic, and4% African American. It was noted that the ethnic diversity variedsignificantly from site to site. For example, one site enrolled 60%Hispanic and 26% Caucasian subjects while three clinics all located inthe same state, enrolled no Hispanic subjects. As expected, there wereno discernible differences observed in our results for differentethnicities.

Training Data Set 1

The training set study selected 71 samples from the initial sequentialaccumulation of 435 samples that were collected between April 2009 andDecember 2009. All subjects with affected fetus' (abnormal karyotypes)in this first series of subjects were included for sequencing and arandom selection and number of non-affected subjects with adequatesample and data. Clinical characteristics of the training set patientswere consistent with the overall study demographics as shown in Table 2.The gestational age range of the samples in the training set ranged from10 weeks, 0 days to 23 weeks 1 day. Thirty-eight underwent CVS, 32underwent amniocentesis and 1 patient did not have the invasiveprocedure type specified (an unaffected karyotype 46, XY). 70% of thepatients were Caucasian, 8.5% Hispanic, 8.5% Asian, and 8.5%multi-ethnic. Six sequenced samples were removed from this set for thepurposes of training: 4 samples from subjects with twin gestations(further discussed below), 1 sample with T18 that was contaminatedduring preparation, and 1 sample with a fetal karyotype 69, XXX, leaving65 samples for the training set.

The number of unique sequence sites (i.e. tags identified with uniquesites in the genome) varied from 2.2M in the early phases of thetraining set study to 13.7M in the latter phases due to improvements insequencing technology over time. In order to monitor for any potentialshifts in the chromosome ratios over this 6-fold range in unique sites,different unaffected samples were run at the beginning and end of thestudy. For the first 15 unaffected samples run, the average number ofunique sites was 3.8M and the average chromosome ratios for chromosome21 and chromosome 18 were 0.314 and 0.528, respectively. For the last 15unaffected samples run, the average number of unique sites was 10.7M andthe average chromosome ratios for chromosome 21 and chromosome 18 were0.316 and 0.529, respectively. There was no statistical differencebetween the chromosome ratios for chromosome 21 and chromosome 18 overthe time of the training set study.

The training set NCVs for chromosomes 21, 18 and 13 are shown on FIG. 2.The results shown in FIG. 2 are consistent with an assumption ofnormality in that roughly 99% of the diploid NCVs would fall within ±2.5standard deviations of the mean. Of this set of 65 samples, 8 sampleswith clinical karyotypes indicating T21 had NCVs ranging from 6 to 20.Four samples having clinical karyotypes indicative of fetal T18 had NCVsranging from 3.3 to 12, and the two samples having karyotypes indicativeof fetal trisomy 13 (T13) had NCVs of 2.6 and 4. The spread of the NCVsin affected samples is due to their dependence on the percentage offetal cfDNA in the individual samples.

Similar to the autosomes, the means and standard deviations for the sexchromosomes were established in the training set. The sex chromosomethresholds allowed 100% identification of male and female fetuses in thetraining set.

Test Data Set 1

Having established chromosome ratio means and standard deviations fromthe training set, a test set of 48 samples was selected from samplescollected between January 2010 and June 2010 from 575 total samples. Oneof the samples from a twin gestation was removed from the final analysisleaving 47 samples in the test set. Personnel preparing samples forsequencing and operating the equipment were blinded to the clinicalkaryotype information. The gestational age range was similar to thatseen in the training set (Table 2). 58% of the invasive procedures wereCVS, higher than that of the overall procedural demographics, but alsosimilar to the training set. 50% of subjects were Caucasian, 27%Hispanic, 10.4% Asian and 6.3% African American.

In the test set, the number of unique sequence tags varied fromapproximately 13M to 26M. For unaffected samples, the chromosome ratiosfor chromosome 21 and chromosome 18 were 0.313 and 0.527, respectively.The test set NCVs for chromosome 21, chromosome 18 and chromosome 13 areshown in FIG. 3 and the classifications are given in Table 3.

TABLE 3 Test Set Classification Data Test Set Classification Data T21classification Unaffected Karyotype for T21 T21 No Call Unaffected forT21 34 47,XX or XY + 21 13 T18 classification Unaffected Karyotype forT18 T18 No Call Unaffected for T18 39 47,XX or XY + 18 8 T13classification Unaffected Karyotype for T13 T13 No Call Unaffected forT13 46 47,XX or XY + 13 1 Sex Chromosome Classification Karyotype XY XXMX* No Call 46, XY 24 46, XX 18 1 45, X 2 1 Cplx 1 *MX is monosomy inthe X chromosome with no evidence of Y chromosome

In the test set, 13/13 subjects having clinical karyotypes thatindicated fetal T21 were correctly identified having NCVs ranging from 5to 14. Eight/eight subjects having karyotypes that indicated fetal T18were correctly identified having NCVs ranging from 8.5 to 22. The singlesample having a karyotype classified as T13 in this test set wasclassified as a no call with an NCV of approximately 3.

For the test data set, all male samples were correctly identifiedincluding a sample with complex karyotype, 46,XY+marker chromosome(unidentifiable by cytogenetics) (Table 3). Nineteen of twenty femalesamples were correctly identified, and one female sample was categorizedas a no call. For three samples in the test set with karyotype of 45,X,two of the three were correctly identified as monosomy X and 1 wasclassified as a no call (Table 3).

Twins

Four of the samples initially selected for the training set and one ofthe samples in the test set were from twin gestations. The thresholdsbeing employed here could be confounded by the differing amount of cfDNAexpected in the setting of a twin gestation. In the training set, thekaryotype from one of the twin samples was monochorionic 47,XY+21. Asecond twin sample was fraternal and amniocentesis was carried out oneach of the fetuses individually. In this twin gestation, one of thefetuses had a karyotype of 47,XY+21 while the other had a normalkaryotype, 46,XX. In both of these cases the cell free classificationbased on the methods discussed above classified the sample as T21. Theother two twin gestations in the training set were classified correctlyas non-affected for T21 (all twins showed diploid karyotype forchromosome 21). For the twin gestation sample in the test set, karyotypewas only established for Twin B (46,XX) and the algorithm correctlyclassified as non-affected for T21.

Conclusion

The data show that massively parallel sequencing can be used todetermine a plurality abnormal fetal karyotypes from the blood ofpregnant women. These data demonstrate that 100% correct classificationof samples with trisomy 21 and trisomy 18 can be identified usingindependent test set data. Even in the case of fetuses with abnormal sexchromosome karyotypes, none of the samples were incorrectly classifiedwith the algorithm of the method. Importantly, the algorithm alsoperformed well in determining the presence of T21 in two sets of twinpregnancies having at least one affected fetus, which has never beenshown previously. Furthermore, this study examined a variety ofsequential samples from multiple centers representing not only the rangeof abnormal karyotypes that one is likely to witness in a commercialclinical setting, but showing the significance of accurately classifyingpregnancies non-affected by common trisomies to address the unacceptablyhigh false positive rates that remain in prenatal screening today. Thedata provide valuable insight into the vast capabilities of employingthis method in the future. Analysis of subsets of the unique genomicsites showed increases in the variance consistent Poisson countingstatistics.

The data build on the findings of Fan and Quake who demonstrated thatthe sensitivity of noninvasive prenatal determination of fetalaneuploidy from maternal plasma using massively parallel sequencing isonly limited by the counting statistics (Fan and Quake, PLos One 5,e10439 [2010]). Because sequencing information was collected across theentire genome, this method is capable of determining any aneuploidy orother copy number variation including insertions and deletions. Thekaryotype from one of the samples had a small deletion in chromosome 11between q21 and q23 that was observed as a ˜10% decrease in the relativenumber of tags in a 25 Mbase region starting at q21 when the sequencingdata was analyzed in 500 kbase bins. In addition, in the training set,three of the samples had complex sex karyotypes due to mosaicism in thecytogenetic analysis. These karyotypes were: i) 47,XXX[9]/45,X[6], ii)45,X [3]/46, XY[17], and iii) 47,XXX[13]/45,X[7]. Sample ii, whichshowed some XY-containing cells was correctly classified as XY. Samplesi (from CVS procedure) and iii (from amniocentesis), which both showed amixture of XXX and X cells by cytogenetic analysis (consistent withmosaic Turner syndrome), were classified as a no call and monosomy X,respectively.

In testing the algorithm, another interesting data point was observedhaving an NCV between −5 and −6 for chromosome 21 for one sample fromthe test set (FIG. 3). Although this sample was diploid in chromosome 21by cytogenetics, the karyotype showed mosaicism with partial triploidyfor chromosome 9; 47, XX+9 [9]/46, XX [6]. Since chromosome 9 is used inthe denominator to determine the chromosome dose for chromosome 21(Table 1), this lowers the overall NCV value. The result strikinglydemonstrates the ability of the method to determine fetal trisomy 9 inthis case (see Example 2). Multiple chromosome ratios were determined toinsure correct classification for the chromosomes of interest. Inaddition, normalizing chromosomes for all the autosomes were establishedto increase the probability of determining rare aneuploidies across thegenome (See Example 5).

The conclusion of Fan, et al regarding the sensitivity of these methodsis only correct if the algorithms being utilized are able to account forany random or systematic biases introduced by the sequencing method. Ifthe sequencing data is not properly normalized the resulting analysiswill be inferior to the counting statistics. Chiu, et al noted in theirrecent paper that their measurement of chromosomes 18 and 13 using themassively parallel sequencing method was imprecise, and concluded thatmore research was necessary to apply the method to the determination ofT18 and T13 (Chiu et al., BMJ 342:c7401 [2011]). The method utilized inthe Chiu, et al paper simply uses the number of sequence tags on thechromosome of interest, in their case chromosome 21, normalized by thetotal number of tags in the sequencing run. The challenge for thisapproach is that the distribution of tags on each chromosome can varyfrom sequencing run to sequencing run, and thus increases the overallvariation of the aneuploidy determination metric. In order to comparethe results of the Chiu algorithm to the chromosome ratios used in thispaper, the test data for chromosomes 21 and 18 was reanalyzed using themethod recommended by Chiu, et al as shown in FIG. 4. Overall, acompression in the range of NCV for each of the chromosomes 21 and 18was observed as well as a decrease in the determination rate with 10/13T21 and 5/8 of the T18 samples correctly identified from our test setutilizing an NCV threshold of 4.0 for aneuploidy classification.

Ehrich, et al also focused only on T21 and used the same algorithm asChiu, et al., (Ehrich et al., Am J Obstet Gynecol 204:205 e1-ell[2011]). In addition, after observing a shift in their test set z-scoremetric from the external reference data i.e. training set, theyretrained on the test set to establish the classification boundaries.Although in principle this approach is feasible, in practice it would bechallenging to decide how many samples are required to train and howoften one would need to retrain to ensure that the classificationboundaries are correct. One method of mitigating this issue is toinclude controls in every sequencing run that measure the baseline andcalibrate for quantitative behavior.

The data obtained using the present method show that massively parallelsequencing is capable of determining multiple fetal chromosomalabnormalities from the plasma of pregnant women when the algorithm fornormalizing the chromosome counting data is optimized. The presentmethod for quantification not only minimizes random and systematicvariations between sequencing runs, but also allow for effectiveclassification of aneuploidies across the entire genome, most notablyT21 and T18. Larger sample collections are required to test thealgorithm for T13 determination. To this end, a prospective, blinded,multi-site clinical study to further demonstrate the diagnostic accuracyof the present method is being performed.

Example 2 Use of Multiple Chromosome Ratios to Verify Determination ofAneuploidy: Normalizing Normalizing Chromosomes

As described in the previous Example, the present method is based on thenormalization of the number sequence tags mapped to a chromosome ofinterest to the number of sequence tags mapped to a chromosome thatdisplays similar variability across samples and across sequencing runsto the chromosome of interest. To verify the classification of ananeuploidy and exclude that the normalizing chromosome used in theanalysis is itself an aneuploid chromosome i.e. present in aberrant copynumber, normalization of the first normalizing chromosome i.e. thechromosome used for determining a chromosome dose for classifying thecommon aneuploidies involving chromosomes 21, 18 and X, was determinedas follows.

Using the qualifying samples from the training set 1, and the qualifyingsamples from test set 1 as described in Example 1, sequencinginformation was analyzed to identify at least one second normalizingchromosome for the first normalizing chromosome used to determine thepresence or absence of a T21, T18 or chromosome X aneuploidy (see Tables4, 5, and 6, respectively).

A. Second Normalizing Chromosome for First Normalizing Chromosome 9:

To verify the determination of a normal chromosome 21 genotypedetermined using first normalizing chromosome 9 as determined in Example1, chromosome doses were calculated for chromosome 9 using each of theother chromosomes i.e. as ratios of tags mapped to chromosome 9 to tagsmapped to chromosomes 1-8, and 10-22 in each of qualified samples(normal samples) in the training set 1, and in each of the qualifiedsamples in the test set, and the % CV was calculated (Table 4). Asdescribed previously, the % CV used to identify normalizing chromosomesare CV values of chromosome doses determined in diploid samples.

TABLE 4 Determination of Second Normalizing Chromosomes for FirstNormalizing Chromosome 9 Training Set 1 Test set 1 % CV % CV Chr229.291074 Chr19 8.111788 Chr19 8.897349 Chr22 7.855368 Chr4 5.76344 Chr45.284925 Chr17 5.571726 Chr17 5.121887 Chr16 4.53673 Chr16 4.190157Chr20 4.058794 Chr20 3.830602 Chr5 3.237778 Chr5 2.825374 Chr6 3.181269Chr6 2.800885 Chr3 2.981951 Chr3 2.67343 Chr8 2.111639 Chr8 1.819142Chr2 1.75712 Chr2 1.680979 Chr7 1.526366 Chr7 1.357402 Chr12 1.328557Chr15 1.311336 Chr15 1.30808 Chr12 1.122218 Chr14 0.999624 Chr140.954458 Chr10 0.770065 Chr1 0.86791 Chr1 0.720795 Chr10 0.781235 Chr110.625072 Chr11 0.611422 Chr9 0 Chr9 0

The chromosome having the lowest variability was determined to bechromosome 11 in qualified samples from both the training set and thetest set.

Having selected chromosome 11 as the second normalizing chromosome forthe verifying the determination of aneuploidy for chromosome 21 i.e.T21, using first normalizing chromosome 9, chromosome doses forchromosome 9/chromosome 11 were calculated for each of the test samples.NCVs for each of the test samples were determined as described inExample 1 using the average chromosome dose of 0.834054±0.005213(mean±S.D.) for chromosome 9/chromosome 11 as determined in thequalified samples of the training set (FIG. 5).

The data show that the aberrantly low NCV calculated for chromosome 21using chromosome 9 (5-6 NCV below the mean of the remaining testsamples; FIG. 3) corresponds with an aberrantly high NCV for chromosome9 when using chromosome 11 as the second normalizing chromosome (5-6 NCVabove the mean of the remaining test samples). The data indicate thatthe sample has a chromosome 9 aneuploidy, and verify the determinationof a diploid chromosome 21 in the sample. This result is consistent withan aneuploid karyotype for the sample, which had been shown to be atrisomy 9 mosaic 47, XX+9 [9]/46, XX [6]. The karyotype of the trisomy 9was determined using an amniotic fluid sample. In addition, these datashow that the method is capable of identifying rare chromosomalaneuploidies e.g. trisomy 9.

B. Second Normalizing Chromosome for First Normalizing Chromosome 8:

Chromosome doses for chromosome 8, which is the normalizing chromosomethat was used to determine the presence or absence of T18 as describedin Example 1, were calculated for each of the other chromosomes i.e. asratios of tags mapped to chromosome 8 to chromosomes 1-7, and 9-22 ineach of qualified samples (normal samples) in training set 1, and ineach of the qualified samples in test set 1, and the % CV was calculated(Table 5).

TABLE 5 Determination of Second Normalizing Chromosomes for FirstNormalizing Chromosome 8 Training set 1 Test set 1 % CV % CV Chr2211.46522 Chr19 9.798657 Chr19 11.047 Chr22 9.563598 Chr17 7.703968 Chr176.814218 Chr16 6.62974 Chr16 5.872925 Chr20 6.141408 Chr20 5.520658 Chr43.705126 Chr4 3.528544 Chr15 3.206262 Chr15 2.863827 Chr10 2.698991 Chr12.340634 Chr1 2.693637 Chr10 2.249778 Chr11 2.519884 Chr11 2.03519 Chr92.117622 Chr9 1.808857 Chr14 1.268471 Chr5 1.046654 Chr5 1.175661 Chr61.042236 Chr6 1.141192 Chr14 1.011047 Chr3 0.962767 Chr3 0.89667 Chr120.902309 Chr12 0.819304 Chr7 0.699651 Chr7 0.605317 Chr2 0.529831 Chr20.304911 Chr8 0 Chr8 0

The chromosome having the lowest variability was determined to bechromosome 11 in qualified samples from both the training set and thetest set.

Having selected chromosome 2 as the second normalizing chromosome forthe verifying the determination of aneuploidy for chromosome 18 i.e.T18, using first normalizing chromosome 8, chromosome doses forchromosome 8/chromosome 2 were calculated for each of the test samples.NCVs for each of the test samples were determined using the averagechromosome dose of 0.60102532±0.00318442 (mean±S.D.) for chromosome8/chromosome 2 as determined in the qualified samples of the trainingset (FIG. 6).

FIG. 6 shows that an aneuploidy for first normalizing chromosome 8 wasabsent in all the test samples, thus verifying the determination of thepresence or absence of a T18 aneuploidy determined using chromosome 8 asthe normalizing chromosome.

C. Second Normalizing Chromosome for First Normalizing Chromosome 6:

Chromosome doses for chromosome 6, which is the normalizing chromosomethat was used to determine the presence or absence of an aneuploidy ofchromosome X as described in Example 1, were calculated for each of theother chromosomes i.e. as ratios of tags mapped to chromosome 6 tochromosomes 1-5, and 7-22 in each of qualified samples (normal samples)in the training set, and in each of the qualified samples in the testset, and the % CV was calculated (Table 6).

TABLE 6 Determination of Second Normalizing Chromosomes for FirstNormalizing Chromosome 6 Training set 1 Test set 1 % CV % CV Chr2212.5071 Chr19 10.78333 Chr19 12.0931 Chr22 10.54754 Chr17 8.746929 Chr177.786613 Chr16 7.688881 Chr16 6.870243 Chr20 7.189759 Chr20 6.517251Chr15 4.216819 Chr15 3.828722 Chr10 3.742112 Chr1 3.301578 Chr1 3.722392Chr10 3.229611 Chr11 3.558687 Chr11 3.025084 Chr9 3.171762 Chr9 2.791694Chr4 2.642087 Chr4 2.563439 Chr14 2.275682 Chr14 1.962283 Chr12 1.905575Chr12 1.774915 Chr7 1.730526 Chr7 1.534558 Chr2 1.461087 Chr2 1.147422Chr8 1.136377 Chr8 1.04368 Chr3 0.367681 Chr5 0.306341 Chr5 0.32993 Chr30.245471 Chr6 0 Chr6 0

The chromosome having the lowest variability was determined to bechromosome 5 in qualified samples in the training set, and chromosome 3in the qualified samples of the test set.

Having selected chromosome 5 as the second normalizing chromosome forverifying the determination of aneuploidy for chromosome X e.g. monosomyX using first normalizing chromosome 6, chromosome doses for chromosome6/chromosome 5 were calculated for each of the test samples. NCVs foreach of the test samples were determined using the average chromosomedose of 0.954309±0.003149 (mean±S.D.) for chromosome 6/chromosome 5 asdetermined in the qualified samples of the training set 1.

FIG. 7 shows that an aneuploidy for second normalizing chromosome 5 wasabsent in all the test samples, thus verifying the determination of thepresence or absence of a chromosome X aneuploidy determined usingchromosome 6 as the first normalizing chromosome.

These data indicate that the present method can be used to determinerare aneuploidies e.g. trisomy 9, and that the present method can beused to verify the result of a determination of the presence or absenceof an aneuploidy for chromosomes of interest by normalizing the firstnormalizing chromosome with a second normalizing chromosome.Normalization of the first normalizing chromosome verifies the firstresults by confirming the presence or absence of an aneuploidy for thefirst normalizing chromosome, and determining the presence or absence ofan aneuploidy in the first or second normalizing chromosome.

Example 3 Determination and Verification of a Chromosomal AneuploidyUsing at Least Two Normalizing Chromosomes for a Chromosome of Interest

To demonstrate that the determination of a chromosomal aneuploidy can beverified by using a first and a second normalizing chromosome for achromosome of interest, the chromosome doses for chromosome 21 inExample 1A that were computed using chromosome 9 as the firstnormalizing chromosome, were calculated using chromosome 10 andchromosome 14 as the second and third normalizing chromosome forchromosome of interest 21.

FIG. 8A shows a plot of NCVs for the 48 samples in test set 1 calculatedusing the mean and S.D. of the corresponding chromosome doses in thequalified samples of training set 1. The mean % CV of chromosome dosesfor chromosome 21 in training set 1 are provided in Table 7.

TABLE 7 Determination of Second Normalizing Chromosomes for Chromosomeof Interest Chromosome 21 Training Set 1 Test Set 1 % CV % CV ChrY81.98064 ChrY 82.78696 Chr22 8.096499 Chr19 8.646585 Chr19 7.657979Chr22 8.445861 ChrX 5.651177 Chr17 5.671588 Chr4 5.408138 ChrX 5.586468Chr17 4.958059 Chr4 4.920301 Chr13 4.65324 Chr16 4.793311 Chr16 4.148073Chr20 4.476581 Chr20 3.803907 Chr13 4.071612 Chr5 3.124174 Chr183.570399 Chr6 3.059323 Chr5 2.398359 Chr3 2.897794 Chr6 2.37894 Chr182.550919 Chr3 2.268173 Chr8 2.052522 Chr15 1.862794 Chr2 1.784604 Chr81.400578 Chr7 1.532462 Chr1 1.3214 Chr12 1.488691 Chr2 1.303358 Chr151.445682 Chr10 1.232134 Chr14 1.212778 Chr11 0.959397 Chr1 1.090117 Chr70.956681 Chr10 1.089907 Chr12 0.851336 Chr11 1.037469 Chr9 0.772917 Chr90.747884 Chr14 0.741307 Chr21 0 Chr21 0

The test sample identified in FIG. 3 for having an unusually low NCV ofbetween −5 and −6 NCV and having been classified correctly as a diploidfor chromosome 21 when using chromosome 9 as a first normalizingchromosome is indicated in FIG. 8A by the arrow. In addition to usingchromosome 9 as the first normalizing chromosome, the presence orabsence of trisomy 21 was determined in all tests samples of test set 1using chromosome 10 and using chromosome 14 as additional normalizingchromosomes. An average of 0.259070±0.002823 S.D. was used for secondnormalizing chromosome 10, and an average 0.409420±00.4965 S.D. was usedfor second normalizing chromosome 14 to calculate the NCVs shown inFIGS. 8B and 8C, respectively.

The data shown in FIGS. 8 B and C show that the sample previouslyclassified as diploid for chromosome 21 when chromosome 9 was used as afirst normalizing chromosome (FIGS. 3 and 8A), was confirmed to bediploid for chromosome 21 when chromosome 10 (FIG. 8B) or chromosome 14(FIG. 8C) were used as the normalizing chromosomes.

Therefore, determination of the presence or absence of a chromosomalaneuploidy can be verified by using at least two different chromosomesas normalizing chromosomes for a chromosome of interest.

Example 4 Determination of a Chromosomal Aneuploidy in SecondNormalizing Chromosome for First Normalizing Chromosome 8

To demonstrate that in addition to determining the presence of rarechromosomal abnormalities other than the trisomy 9 as determined inExamples 1 and 2, sequence information was obtained from a secondtraining set and a second test set, and NCVs for all chromosome dosesfor each of chromosomes 1-22 were calculated as described above.

Determinations of the presence or absence of aneuploidies involvingchromosome 18 in samples from Test set 2 were made using chromosome 8 asthe first normalizing chromosome. To verify that the determinations ofthe presence or absence of trisomy 18 in the test samples, chromosomedoses for chromosome 8 were calculated for each of the other chromosomesi.e. as ratios of tags mapped to chromosome 8 to chromosomes 1-7, and9-22 in each of qualified samples (normal samples) in training set 2,and in each of the qualified samples in test set 2, and the % CV wascalculated (Table 8).

TABLE 8 Determination of Second Normalizing Chromosomes for FirstNormalizing Chromosome 8 Training set 2 Test set 2 % CV % CV Chr195.904815 Chr22 8.770626 Chr22 5.556697 Chr19 8.562072 Chr17 4.25183Chr17 6.237912 Chr16 3.279849 Chr16 4.900381 Chr20 3.077099 Chr204.681362 Chr15 2.022277 Chr4 2.787432 Chr4 1.717953 Chr15 2.565708 Chr11.631726 Chr1 2.305568 Chr11 1.400395 Chr11 2.024559 Chr10 1.371933Chr10 2.022278 Chr9 1.234463 Chr9 1.757622 Chr14 0.899747 Chr14 1.163575Chr12 0.733874 Chr6 0.860914 Chr7 0.66061 Chr5 0.852043 Chr6 0.520435Chr7 0.818984 Chr5 0.502611 Chr12 0.801362 Chr3 0.484482 Chr3 0.764795Chr2 0.400574 Chr2 0.525098 Chr8 0 Chr8 0

The chromosome having the lowest variability was determined to bechromosome 2 in qualified samples from both the training set and thetest set, and was use as the second normalizing chromosome for verifyingthe determination of the presence or absence of an aneuploidy forchromosome 18. Using first normalizing chromosome 8, chromosome dosesfor chromosome 8/chromosome 2 were calculated for each of the testsamples. NCVs for each of the test samples were determined using theaverage chromosome dose of 0.601163±0.002408 (mean±S.D.) for chromosome8/chromosome 2 as determined in the qualified samples of training set 2(FIG. 9A). FIG. 9A shows an aneuploidy in a test sample that wasanalyzed for T18 using first normalizing chromosome 8. The abnormallylow NCV of about −10 for the chromosome 8 dose when using chromosome 2as the second normalizing chromosome indicates the presence of ananeuploidy for chromosome 2 in the test sample. To verify that theaneuploidy rests with chromosome 2 and not chromosome 8, NCVs for eachof the test samples were determined using the average chromosome dose of0.953953±0.006302 (mean±S.D.) for chromosome 8/chromosome 7 asdetermined in the qualified samples of training set 2 (FIG. 9B). FIG. 9Bshows that none of the test samples contained an aneuploid chromosome 8when chromosome 7 was used as second normalizing chromosome to calculatedoses and NCVs for first normalizing chromosome 8.

These data confirm that the present method can be used to determine rareaneuploidies, and that the present method can be used to verify theresult of a determination of the presence or absence of an aneuploidy bydetermining that the first normalizing chromosome used as the numeratorused to calculate the dose of a chromosome of interest is itself notpresent in aberrant copy numbers i.e. it is not an aneuploid chromosome.As shown in Examples 2 and 3, the determination of the presence orabsence of an aneuploidy can be made by using at least two differentnormalizing chromosomes. The different normalizing chromosomes can beused as separate numerators when calculating the chromosome dose and NCVfor a chromosome of interest, and comparing the results to ascertain thesame outcome. Alternatively, the first of the two different normalizingchromosomes can be used to calculate the dose and NCV for a chromosomeof interest, and the second normalizing chromosome can be used tocalculate the dose and NCV of the first normalizing chromosome to verifythat the first normalizing chromosome is devoid of an aneuploidy.

Example 5 Determination of First and Second Normalizing Chromosomes forthe Determination of Chromosomal Aneuploidies

To identify normalizing chromosomes for each of chromosomes 1-2, X andY, sequencing information obtained from sequencing all samples i.e.qualified and affected, from each of training set 1, test set 1, andtraining set 2, was used to compute percent NCVs for each chromosomeusing all chromosomes as described in the previous Examples.

The data shown in Table 9, provides four normalizing chromosomes foreach of all 1-22, X and Y chromosomes that were determined to have thelowest CVs for the respective doses in the 3 sample sets provided.

Normalizing chromosomes having the lowest four % CVs are provided. Thesecond lowest variability for chromosome 13 was determined to resultfrom the average of the sum of chromosome doses for chromosomes 2-6. Thevariability of the chromosome dose for chromosome Y is smallest when theaverage of the sum of chromosome doses for chromosomes 2-6 is used.

TABLE 9 Normalizing Chromosomes for all Chromosomes NormalizingNormalizing Normalizing chromosomes - chromosomes - chromosomes -Training set 1 Test set 1 Training set 2 Chromosome n = 65 n = 48 n = 48 1 11, 10, 9, 15 11, 10, 15, 9 10, 11, 9, 15  2 8, 12, 18, 7 8, 12, 7,14 7, 8, 12, 14  3 6, 5, 18, 8 6, 5, 8, 18 6, 5, 8, 18  4 13, 5, 3, 613, 5, 6, 3, 13, 5, 6, 3  5 3, 6, 18, 8 3, 6, 8, 18 6, 3, 8, 18  6 3, 5,18, 8 3, 5, 8, 18 5, 3, 8, 18  7 12, 14, 2, 8 12, 14, 2, 8 12, 2, 8, 14 8 2, 3, 5, 6, 2, 3, 12, 7 2, 7, 12, 3  9 1, 10, 11, 7 11, 10, 1, 14 11,1, 10, 14 10 11, 9, 1, 14 11, 1, 9, 15 1, 11, 9, 15 11 9, 10, 1, 21 9,10, 1, 15 1, 10, 9, 15 12 14, 7, 2, 9, 7, 14, 2, 8 7, 14, 2, 8 13 4,2-6, 5, 6 4, 2-6, 5, 6 4, 5, 2-6, 3 14 12, 7, 9, 21 12, 7, 9, 2 12, 7,2, 9 15 1, 11, 10, 9 1, 10, 11, 9, 1, 10, 11, 9, 16 20, 17, 15, 1 20,17, 15, 1 20, 17, 15, 10 17 16, 20, 22, 19 12, 20, 19, 22 16, 20, 19, 2218 8, 3, 6, 2, 8, 5, 6, 8 8, 3, 2, 6 19 22, 17, 16, 20 22, 17, 16, 2022, 17, 16, 20 20 16, 17, 15, 1 16, 17, 15, 1 16, 17, 15, 10 21 9, 11,10, 1 14, 9, 12, 7 14, 9, 11, 7 22 19, 17, 16, 20 19, 17, 16, 20 19, 17,16, 20 X 4, 13, 5, 3 4, 13, 5, 3 5, 13, 3, 6 Y 2-6, 4, 7, 5 2-6, 13, 5,4 2-6, 5, 4, 3

Based on the results, normalizing chromosomes can be selected whetherthe second normalizing chromosome is one of two selected normalizingchromosomes for a chromosome of interest, or the second normalizingchromosome is a normalizing chromosome for the first normalizingchromosome, which is the first normalizing chromosome for a chromosomeof interest.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for determining the presence or absence of a fetal chromosomal aneuploidy in a maternal test sample obtained from a pregnant human, said sample comprising a mixture of fetal and maternal nucleic acid molecules, said method comprising: (a) preparing a sequencing library from said mixture of fetal and maternal nucleic acids in said maternal sample; (b) massively parallel sequencing said fetal and maternal nucleic acids in said sequencing library to obtain sequence information for said fetal and maternal nucleic acids in said sample; (c) receiving said sequence information in a computer-readable medium; (d) using computer-readable instructions: (i) identifying from said sequence information a number of sequence tags for a chromosome of interest and a number of sequence tags for at least two normalizing chromosomes; (ii) using said numbers of sequence tags to calculate a first normalizing value and a second normalizing value for said chromosome of interest; and (iii) comparing said first normalizing value for said chromosome of interest to a first threshold value and comparing said second normalizing value for said chromosome of interest to a second threshold value to determine the presence or absence of a fetal aneuploidy in said sample.
 2. The method of claim 1, wherein said first normalizing value for said chromosome of interest is a first chromosome dose, said first chromosome dose being a ratio of the number of sequence tags for said chromosome of interest and a first normalizing chromosome, and wherein said second normalizing value for said chromosome of interest is a second chromosome dose, said second chromosome dose being a ratio of the number of sequence tags for said chromosome of interest and a second normalizing chromosome.
 3. A method for determining the presence or absence of a fetal chromosomal aneuploidy in a maternal test sample obtained from a pregnant human, said sample comprising a mixture of fetal and maternal nucleic acid molecules, said method comprising: (a) preparing a sequencing library from said mixture of fetal and maternal nucleic acids in said maternal sample; (b) massively parallel sequencing said fetal and maternal nucleic acids in said sequencing library to obtain sequence information for said fetal and maternal nucleic acids in said sample; (c) receiving said sequence information in a computer-readable medium; (d) using computer-readable instructions: (i) identifying from said sequence information a number of sequence tags for a chromosome of interest and a number of sequence tags for at least two normalizing chromosomes; (ii) using said number of sequence tags for said chromosome of interest and the number of sequence tags for a first normalizing chromosome for said chromosome of interest to determine a first normalizing value for said chromosome of interest, and using said number of sequence tags for said first normalizing chromosome and the number of sequence tags for a second normalizing chromosome to determine a second normalizing value for said first normalizing chromosome; and (iii) comparing said first normalizing value for said chromosome of interest to a first threshold value and comparing said second normalizing value for said first normalizing chromosome to a second threshold value to determine the presence or absence of a fetal aneuploidy in said sample.
 4. The method of claim 3, wherein said first normalizing value for said chromosome of interest is a first chromosome dose, said first chromosome dose being a ratio of the number of sequence tags for said chromosome of interest and a first normalizing chromosome, and wherein said second normalizing value for said chromosome of interest is a second chromosome dose, said second chromosome dose being a ratio of the number of sequence tags for said first normalizing chromosome and a second normalizing chromosome.
 5. The method of any one of claim 1 or claim 3, further comprising determining a first and a second normalized chromosome value (NCV), wherein said first NCV relates said first chromosome dose to the mean of the corresponding first chromosome dose in a set of qualified samples, and wherein said second NCV relates said second chromosome dose to the mean of the corresponding second chromosome dose in a set of qualified samples as: ${NCV}_{ij} = \frac{x_{ij} - {\hat{\mu}}_{j}}{{\hat{\sigma}}_{j}}$ where {circumflex over (μ)}_(j) AND {circumflex over (σ)}_(j) are the estimated mean and standard deviation, respectively, for the j-th chromosome dose in a set of qualified samples, and x_(ij) is the observed j-th chromosome dose for test sample i.
 6. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 21, and said normalizing chromosomes are selected from chromosomes 9, 11, 14, and
 1. 7. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 18, and said normalizing chromosomes are selected from chromosomes 8, 3, 2, and
 6. 8. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 13, and said normalizing chromosomes are selected from chromosome 4, the group of chromosomes 2-6, chromosome 5, and chromosome
 6. 9. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome X, and said normalizing chromosomes are selected from chromosomes 6, 5, 13, and
 3. 10. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 1, and said normalizing chromosomes are selected from chromosomes 10, 11, 9 and
 15. 11. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 2, and said normalizing chromosomes are selected from chromosomes 8, 7, 12, and
 14. 12. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 3, and said normalizing chromosomes are selected from chromosomes 6, 5, 8, and
 18. 13. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 4, and said normalizing chromosomes are selected from chromosomes 3, 5, 6, and
 13. 14. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 5, and said normalizing chromosomes are selected from chromosomes 6, 3, 8, and
 18. 15. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 6, and said normalizing chromosomes are selected from chromosomes 5, 3, 8, and
 18. 16. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 7, and said normalizing chromosomes are selected from chromosomes 12, 2, 14 and
 8. 17. The method of any one of claim 1 or claim 3, wherein: said normalizing chromosomes are used to normalize chromosome 8, and said normalizing chromosomes are selected from chromosomes 2, 7, 12, and
 3. 18. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 9, and said normalizing chromosomes for chromosome 9 are selected from chromosomes 11, 10, 1, and
 14. 19. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 10, and said normalizing chromosomes are selected from chromosomes 1, 11, 9, and
 15. 20. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 11, and said normalizing chromosomes are selected from chromosomes 1, 10, 9, and
 15. 21. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 12, and said normalizing chromosomes are selected from chromosomes 7, 14, 2, and
 8. 22. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 14, and said normalizing chromosomes are selected from chromosomes 12, 7, 2, and
 9. 23. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 15, and said normalizing chromosomes are selected from chromosomes 1, 10, 11, and
 9. 24. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 16, and said normalizing chromosomes are selected from chromosomes 20, 17, 15, and
 1. 25. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 17, and said normalizing chromosomes are selected from chromosomes 16, 20, 19 and
 22. 26. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 19, and said normalizing chromosomes are selected from 22, 17, 16, and
 20. 27. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 20, and said normalizing chromosomes are selected from chromosomes 16, 17, 15, and
 1. 28. The method of any one of claim 1 or claim 3, wherein said normalizing chromosomes are used to normalize chromosome 22, and said normalizing chromosomes are selected from chromosomes 19, 17, 16, and
 20. 29. The method of any one of claim 1 or claim 3, further comprising repeating the steps of claim 1 or claim 3 for at least two chromosomes of interest to determine the presence or absence of said different fetal chromosomal aneuploidies.
 30. The method of claim 29, wherein the method comprises repeating the method of claim 1 or claim 3 for all chromosomes to determine the presence or absence of different fetal chromosomal aneuploidies.
 31. The method of claim 1 or claim 3, wherein said fetal chromosomal aneuploidy is selected from T21, T13, T18, and monosomy X.
 32. The method of claim 1 or claim 3, wherein obtaining sequence information for the fetal and maternal nucleic acids in the sample comprises sequencing fetal and maternal nucleic acid molecules in the sample.
 33. The method of claim 1 or claim 3, wherein: obtaining said sequence information comprises sequencing-by-synthesis using reversible dye terminators; obtaining said sequence information comprises sequencing-by-ligation; or obtaining said sequence information comprises single molecule sequencing.
 34. The method of claim 1 or claim 3, wherein said chromosomal aneuploidy is a partial or complete chromosomal aneuploidy.
 35. The method of claim 1 or claim 3, wherein said maternal test sample is a plasma sample obtained from a pregnant woman and said nucleic acid molecules are cfDNA molecules. 